US20080038717A1 - Enrichment method for variant proteins with altered binding properties - Google Patents
Enrichment method for variant proteins with altered binding properties Download PDFInfo
- Publication number
- US20080038717A1 US20080038717A1 US11/761,180 US76118007A US2008038717A1 US 20080038717 A1 US20080038717 A1 US 20080038717A1 US 76118007 A US76118007 A US 76118007A US 2008038717 A1 US2008038717 A1 US 2008038717A1
- Authority
- US
- United States
- Prior art keywords
- hgh
- protein
- phagemid
- gene
- phage
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 308
- 230000027455 binding Effects 0.000 title claims abstract description 192
- 238000000034 method Methods 0.000 title claims abstract description 140
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 113
- 108010000521 Human Growth Hormone Proteins 0.000 claims abstract description 308
- 102000002265 Human Growth Hormone Human genes 0.000 claims abstract description 296
- 239000000854 Human Growth Hormone Substances 0.000 claims abstract description 295
- 239000002245 particle Substances 0.000 claims abstract description 131
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 58
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 58
- 230000004927 fusion Effects 0.000 claims abstract description 54
- 101710132601 Capsid protein Proteins 0.000 claims abstract description 35
- 101710094648 Coat protein Proteins 0.000 claims abstract description 35
- 101710125418 Major capsid protein Proteins 0.000 claims abstract description 35
- 101710141454 Nucleoprotein Proteins 0.000 claims abstract description 35
- 101710083689 Probable capsid protein Proteins 0.000 claims abstract description 35
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 claims abstract description 34
- 239000003446 ligand Substances 0.000 claims abstract description 19
- 239000013604 expression vector Substances 0.000 claims abstract description 16
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 114
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 107
- 229920001184 polypeptide Polymers 0.000 claims description 93
- 239000013612 plasmid Substances 0.000 claims description 77
- 150000001413 amino acids Chemical class 0.000 claims description 74
- 108020004705 Codon Proteins 0.000 claims description 64
- 239000013598 vector Substances 0.000 claims description 59
- 229910052757 nitrogen Inorganic materials 0.000 claims description 56
- 230000035772 mutation Effects 0.000 claims description 48
- 239000012634 fragment Substances 0.000 claims description 47
- 241000588724 Escherichia coli Species 0.000 claims description 39
- 229940088597 hormone Drugs 0.000 claims description 38
- 239000005556 hormone Substances 0.000 claims description 38
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 19
- 108020005038 Terminator Codon Proteins 0.000 claims description 19
- 230000001105 regulatory effect Effects 0.000 claims description 19
- 125000000539 amino acid group Chemical group 0.000 claims description 15
- 238000013518 transcription Methods 0.000 claims description 15
- 230000035897 transcription Effects 0.000 claims description 15
- 239000000427 antigen Substances 0.000 claims description 13
- 239000003102 growth factor Substances 0.000 claims description 13
- 102000011923 Thyrotropin Human genes 0.000 claims description 12
- 108010061174 Thyrotropin Proteins 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 11
- 108091007433 antigens Proteins 0.000 claims description 11
- 230000001131 transforming effect Effects 0.000 claims description 11
- -1 CD-4 Proteins 0.000 claims description 10
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 10
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 10
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 102000004877 Insulin Human genes 0.000 claims description 9
- 108090001061 Insulin Proteins 0.000 claims description 9
- 102000007644 Colony-Stimulating Factors Human genes 0.000 claims description 8
- 108010071942 Colony-Stimulating Factors Proteins 0.000 claims description 8
- 108010063738 Interleukins Proteins 0.000 claims description 8
- 102000015696 Interleukins Human genes 0.000 claims description 8
- 108091005804 Peptidases Proteins 0.000 claims description 8
- 229940047120 colony stimulating factors Drugs 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 229940047122 interleukins Drugs 0.000 claims description 8
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 7
- 239000004365 Protease Substances 0.000 claims description 7
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 7
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 7
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 7
- 241001524679 Escherichia virus M13 Species 0.000 claims description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 6
- 108010009583 Transforming Growth Factors Proteins 0.000 claims description 6
- 102000009618 Transforming Growth Factors Human genes 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 229910052727 yttrium Inorganic materials 0.000 claims description 6
- 102000002723 Atrial Natriuretic Factor Human genes 0.000 claims description 5
- 101800001288 Atrial natriuretic factor Proteins 0.000 claims description 5
- 108090000204 Dipeptidase 1 Proteins 0.000 claims description 5
- 102000017357 Glycoprotein hormone receptor Human genes 0.000 claims description 5
- 108050005395 Glycoprotein hormone receptor Proteins 0.000 claims description 5
- 108060003951 Immunoglobulin Proteins 0.000 claims description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 5
- 102100036836 Natriuretic peptides B Human genes 0.000 claims description 5
- 101710187802 Natriuretic peptides B Proteins 0.000 claims description 5
- 108010006025 bovine growth hormone Proteins 0.000 claims description 5
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical class C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 claims description 5
- 102000018358 immunoglobulin Human genes 0.000 claims description 5
- 229940072221 immunoglobulins Drugs 0.000 claims description 5
- 102000006495 integrins Human genes 0.000 claims description 5
- 108010044426 integrins Proteins 0.000 claims description 5
- 229940066294 lung surfactant Drugs 0.000 claims description 5
- 239000003580 lung surfactant Substances 0.000 claims description 5
- 108020004999 messenger RNA Proteins 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 5
- 108010059616 Activins Proteins 0.000 claims description 4
- 102000005606 Activins Human genes 0.000 claims description 4
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 claims description 4
- 102000013585 Bombesin Human genes 0.000 claims description 4
- 108010051479 Bombesin Proteins 0.000 claims description 4
- 108010009575 CD55 Antigens Proteins 0.000 claims description 4
- 102000055006 Calcitonin Human genes 0.000 claims description 4
- 108060001064 Calcitonin Proteins 0.000 claims description 4
- 102100022641 Coagulation factor IX Human genes 0.000 claims description 4
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 4
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 claims description 4
- 102000003951 Erythropoietin Human genes 0.000 claims description 4
- 108090000394 Erythropoietin Proteins 0.000 claims description 4
- 108010076282 Factor IX Proteins 0.000 claims description 4
- 108010054218 Factor VIII Proteins 0.000 claims description 4
- 102000001690 Factor VIII Human genes 0.000 claims description 4
- 102400000321 Glucagon Human genes 0.000 claims description 4
- 108060003199 Glucagon Proteins 0.000 claims description 4
- 102000006771 Gonadotropins Human genes 0.000 claims description 4
- 108010086677 Gonadotropins Proteins 0.000 claims description 4
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 4
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims description 4
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 4
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 4
- 101000801481 Homo sapiens Tissue-type plasminogen activator Proteins 0.000 claims description 4
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 4
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 4
- 108010004250 Inhibins Proteins 0.000 claims description 4
- 102000002746 Inhibins Human genes 0.000 claims description 4
- 102000006992 Interferon-alpha Human genes 0.000 claims description 4
- 108010047761 Interferon-alpha Proteins 0.000 claims description 4
- 108090000467 Interferon-beta Proteins 0.000 claims description 4
- 102000003996 Interferon-beta Human genes 0.000 claims description 4
- 102000008070 Interferon-gamma Human genes 0.000 claims description 4
- 108010074328 Interferon-gamma Proteins 0.000 claims description 4
- 108010002352 Interleukin-1 Proteins 0.000 claims description 4
- 108010002350 Interleukin-2 Proteins 0.000 claims description 4
- 108010002386 Interleukin-3 Proteins 0.000 claims description 4
- 108090000978 Interleukin-4 Proteins 0.000 claims description 4
- 102400000401 Latency-associated peptide Human genes 0.000 claims description 4
- 101800001155 Latency-associated peptide Proteins 0.000 claims description 4
- 108090000542 Lymphotoxin-alpha Proteins 0.000 claims description 4
- 102000004083 Lymphotoxin-alpha Human genes 0.000 claims description 4
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 4
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 4
- 102000003729 Neprilysin Human genes 0.000 claims description 4
- 108090000028 Neprilysin Proteins 0.000 claims description 4
- 102000003982 Parathyroid hormone Human genes 0.000 claims description 4
- 108090000445 Parathyroid hormone Proteins 0.000 claims description 4
- 108010076181 Proinsulin Proteins 0.000 claims description 4
- 101800000074 Relaxin A chain Proteins 0.000 claims description 4
- 102400000834 Relaxin A chain Human genes 0.000 claims description 4
- 102400000610 Relaxin B chain Human genes 0.000 claims description 4
- 101710109558 Relaxin B chain Proteins 0.000 claims description 4
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 4
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 4
- 108090000190 Thrombin Proteins 0.000 claims description 4
- 108010000499 Thromboplastin Proteins 0.000 claims description 4
- 102000002262 Thromboplastin Human genes 0.000 claims description 4
- 102100033571 Tissue-type plasminogen activator Human genes 0.000 claims description 4
- 108050006955 Tissue-type plasminogen activator Proteins 0.000 claims description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 4
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 4
- 101800004564 Transforming growth factor alpha Proteins 0.000 claims description 4
- 102400001320 Transforming growth factor alpha Human genes 0.000 claims description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 4
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims description 4
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 claims description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 4
- 239000000488 activin Substances 0.000 claims description 4
- 239000000868 anti-mullerian hormone Substances 0.000 claims description 4
- 102000006635 beta-lactamase Human genes 0.000 claims description 4
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 claims description 4
- 229960004015 calcitonin Drugs 0.000 claims description 4
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 4
- 229940105423 erythropoietin Drugs 0.000 claims description 4
- 229960004222 factor ix Drugs 0.000 claims description 4
- 229960000301 factor viii Drugs 0.000 claims description 4
- 229960004666 glucagon Drugs 0.000 claims description 4
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 4
- 239000002622 gonadotropin Substances 0.000 claims description 4
- 230000002607 hemopoietic effect Effects 0.000 claims description 4
- 239000000893 inhibin Substances 0.000 claims description 4
- 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 claims description 4
- 108091022911 insulin-like growth factor binding Proteins 0.000 claims description 4
- 102000028416 insulin-like growth factor binding Human genes 0.000 claims description 4
- 229910052740 iodine Inorganic materials 0.000 claims description 4
- 230000002138 osteoinductive effect Effects 0.000 claims description 4
- 239000000199 parathyroid hormone Substances 0.000 claims description 4
- 229960001319 parathyroid hormone Drugs 0.000 claims description 4
- 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 claims description 4
- 108010087851 prorelaxin Proteins 0.000 claims description 4
- 230000002441 reversible effect Effects 0.000 claims description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 4
- 229960004072 thrombin Drugs 0.000 claims description 4
- 229940034208 thyroxine Drugs 0.000 claims description 4
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- 229960005356 urokinase Drugs 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 3
- 101710091045 Envelope protein Proteins 0.000 claims description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 3
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims description 3
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 claims description 3
- 102400000022 Insulin-like growth factor II Human genes 0.000 claims description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 3
- 101710188315 Protein X Proteins 0.000 claims description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 3
- 108010023197 Streptokinase Proteins 0.000 claims description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 3
- 230000002608 insulinlike Effects 0.000 claims description 3
- 230000003248 secreting effect Effects 0.000 claims description 3
- 229960005202 streptokinase Drugs 0.000 claims description 3
- 241000701832 Enterobacteria phage T3 Species 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- 229910052698 phosphorus Inorganic materials 0.000 claims description 2
- 229910052721 tungsten Inorganic materials 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims 43
- 229910052805 deuterium Inorganic materials 0.000 claims 13
- 229910052722 tritium Inorganic materials 0.000 claims 13
- 229910052739 hydrogen Inorganic materials 0.000 claims 10
- 101000642577 Homo sapiens Growth hormone variant Proteins 0.000 claims 3
- 102000003839 Human Proteins Human genes 0.000 claims 2
- 108090000144 Human Proteins Proteins 0.000 claims 2
- 102000005763 Thrombopoietin Receptors Human genes 0.000 claims 2
- 108010070774 Thrombopoietin Receptors Proteins 0.000 claims 2
- 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 claims 2
- 102000038455 IGF Type 1 Receptor Human genes 0.000 claims 1
- 108010031794 IGF Type 1 Receptor Proteins 0.000 claims 1
- 102000038460 IGF Type 2 Receptor Human genes 0.000 claims 1
- 108010031792 IGF Type 2 Receptor Proteins 0.000 claims 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims 1
- 102000003743 Relaxin Human genes 0.000 claims 1
- 108090000103 Relaxin Proteins 0.000 claims 1
- 230000003301 hydrolyzing effect Effects 0.000 claims 1
- 229940125396 insulin Drugs 0.000 claims 1
- 239000000122 growth hormone Substances 0.000 abstract description 14
- 102000018997 Growth Hormone Human genes 0.000 abstract description 13
- 108010051696 Growth Hormone Proteins 0.000 abstract description 13
- 238000012216 screening Methods 0.000 abstract description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 5
- 241000724791 Filamentous phage Species 0.000 abstract description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 abstract description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 abstract description 2
- 229940000406 drug candidate Drugs 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 119
- 210000004027 cell Anatomy 0.000 description 96
- 239000011324 bead Substances 0.000 description 92
- 235000018102 proteins Nutrition 0.000 description 80
- 235000001014 amino acid Nutrition 0.000 description 68
- 229940024606 amino acid Drugs 0.000 description 59
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 55
- 239000000872 buffer Substances 0.000 description 48
- 108091034117 Oligonucleotide Proteins 0.000 description 44
- 238000010828 elution Methods 0.000 description 40
- 239000000758 substrate Substances 0.000 description 34
- 102000005962 receptors Human genes 0.000 description 33
- 108020003175 receptors Proteins 0.000 description 33
- 238000006467 substitution reaction Methods 0.000 description 29
- 239000004471 Glycine Substances 0.000 description 26
- 238000002703 mutagenesis Methods 0.000 description 25
- 231100000350 mutagenesis Toxicity 0.000 description 25
- 102000053602 DNA Human genes 0.000 description 23
- 102220513486 Rab-interacting lysosomal protein_E174S_mutation Human genes 0.000 description 22
- 102220318714 rs745780898 Human genes 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 230000000694 effects Effects 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 20
- 229940088598 enzyme Drugs 0.000 description 20
- 239000000203 mixture Substances 0.000 description 20
- 108090000790 Enzymes Proteins 0.000 description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 125000003275 alpha amino acid group Chemical group 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 18
- 208000015181 infectious disease Diseases 0.000 description 17
- 108091008146 restriction endonucleases Proteins 0.000 description 17
- 229960003669 carbenicillin Drugs 0.000 description 16
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 16
- 238000010276 construction Methods 0.000 description 16
- 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 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- 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 14
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 14
- 102000014914 Carrier Proteins Human genes 0.000 description 13
- 102220475746 Probable ATP-dependent RNA helicase DDX6_R64A_mutation Human genes 0.000 description 13
- 239000007983 Tris buffer Substances 0.000 description 12
- 108091008324 binding proteins Proteins 0.000 description 12
- 230000029087 digestion Effects 0.000 description 12
- 238000011534 incubation Methods 0.000 description 12
- 230000003993 interaction Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 11
- 230000008569 process Effects 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 10
- 229920001213 Polysorbate 20 Polymers 0.000 description 10
- 238000012219 cassette mutagenesis Methods 0.000 description 10
- 239000011159 matrix material Substances 0.000 description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108090000787 Subtilisin Proteins 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 239000011230 binding agent Substances 0.000 description 8
- 230000001332 colony forming effect Effects 0.000 description 8
- 231100000219 mutagenic Toxicity 0.000 description 8
- 230000003505 mutagenic effect Effects 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 8
- 230000036964 tight binding Effects 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 108010002519 Prolactin Receptors Proteins 0.000 description 7
- 102100029000 Prolactin receptor Human genes 0.000 description 7
- 235000004279 alanine Nutrition 0.000 description 7
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 7
- 230000002860 competitive effect Effects 0.000 description 7
- 230000001976 improved effect Effects 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 229920002401 polyacrylamide Polymers 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 6
- 102000004594 DNA Polymerase I Human genes 0.000 description 6
- 108010017826 DNA Polymerase I Proteins 0.000 description 6
- 238000001712 DNA sequencing Methods 0.000 description 6
- 102100020948 Growth hormone receptor Human genes 0.000 description 6
- 108010068542 Somatotropin Receptors Proteins 0.000 description 6
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 6
- 230000000996 additive effect Effects 0.000 description 6
- 230000004075 alteration Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 239000000356 contaminant Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000002741 site-directed mutagenesis Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 229910052725 zinc Inorganic materials 0.000 description 6
- 239000011701 zinc Substances 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 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
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 5
- 102000003960 Ligases Human genes 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000013522 chelant Substances 0.000 description 5
- 230000009137 competitive binding Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000006073 displacement reaction Methods 0.000 description 5
- 238000004520 electroporation Methods 0.000 description 5
- 230000037433 frameshift Effects 0.000 description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000004091 panning Methods 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 238000007747 plating Methods 0.000 description 5
- 230000000644 propagated effect Effects 0.000 description 5
- 238000002708 random mutagenesis Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 102220075169 rs763217060 Human genes 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- 210000002845 virion Anatomy 0.000 description 5
- 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 4
- 108010039627 Aprotinin Proteins 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108010016626 Dipeptides Proteins 0.000 description 4
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 4
- 108091027305 Heteroduplex Proteins 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- 108010054278 Lac Repressors Proteins 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 101710163270 Nuclease Proteins 0.000 description 4
- 102000015731 Peptide Hormones Human genes 0.000 description 4
- 108010038988 Peptide Hormones Proteins 0.000 description 4
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 239000004098 Tetracycline Substances 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000000813 peptide hormone Substances 0.000 description 4
- 101150009573 phoA gene Proteins 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 238000010187 selection method Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 229960002180 tetracycline Drugs 0.000 description 4
- 229930101283 tetracycline Natural products 0.000 description 4
- 235000019364 tetracycline Nutrition 0.000 description 4
- 150000003522 tetracyclines Chemical class 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 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
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102220587327 NEDD8-activating enzyme E1 catalytic subunit_H21N_mutation Human genes 0.000 description 3
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 3
- 102220599125 Ras-related protein Rab-1B_D171S_mutation Human genes 0.000 description 3
- 102000009661 Repressor Proteins Human genes 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 238000012867 alanine scanning Methods 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 239000005547 deoxyribonucleotide Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000009510 drug design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000000670 limiting effect Effects 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
- 230000000813 microbial effect Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000013391 scatchard analysis Methods 0.000 description 3
- 238000013207 serial dilution Methods 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000009897 systematic effect Effects 0.000 description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 3
- 229940033663 thimerosal Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000010361 transduction Methods 0.000 description 3
- 230000026683 transduction Effects 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 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 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 102400000739 Corticotropin Human genes 0.000 description 2
- 101800000414 Corticotropin Proteins 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 101000925646 Enterobacteria phage T4 Endolysin Proteins 0.000 description 2
- 241000701533 Escherichia virus T4 Species 0.000 description 2
- 101000643253 Gallus gallus Ribonuclease homolog Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102220465832 La-related protein 1_F10A_mutation Human genes 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108010067902 Peptide Library Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 108010057464 Prolactin Proteins 0.000 description 2
- 102000003946 Prolactin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 108010041111 Thrombopoietin Proteins 0.000 description 2
- 102000036693 Thrombopoietin Human genes 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 230000003305 autocrine Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 229960000258 corticotropin Drugs 0.000 description 2
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000001904 diabetogenic effect Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 230000006651 lactation Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 108700010839 phage proteins Proteins 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 229940097325 prolactin Drugs 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000037432 silent mutation Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940043263 traditional drug Drugs 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PKTAYNJCGHSPDR-JNYFXXDFSA-N (2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-1-[(1R,4S,5aS,7S,10S,11aS,13S,17aS,19R,20aS,22S,23aS,25S,26aS,28S,34S,40S,43S,46R,51R,54R,60S,63S,66S,69S,72S,75S,78S,81S,84S,87S,90S,93R,96S,99S)-51-[[(2S,3R)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-3-hydroxybutanoyl]amino]-81,87-bis(2-amino-2-oxoethyl)-84-benzyl-22,25,26a,28,66-pentakis[(2S)-butan-2-yl]-75,96-bis(3-carbamimidamidopropyl)-20a-(2-carboxyethyl)-7,11a,13,43-tetrakis[(1R)-1-hydroxyethyl]-63,78-bis(hydroxymethyl)-10-[(4-hydroxyphenyl)methyl]-4,23a,40,72-tetramethyl-99-(2-methylpropyl)-a,2,5,6a,8,9a,11,12a,14,17,18a,20,21a,23,24a,26,27a,29,35,38,41,44,52,55,61,64,67,70,73,76,79,82,85,88,91,94,97-heptatriacontaoxo-69,90-di(propan-2-yl)-30a,31a,34a,35a,48,49-hexathia-1a,3,6,7a,9,10a,12,13a,15,18,19a,21,22a,24,25a,27,28a,30,36,39,42,45,53,56,62,65,68,71,74,77,80,83,86,89,92,95,98-heptatriacontazaheptacyclo[91.35.4.419,54.030,34.056,60.0101,105.0113,117]hexatriacontahectane-46-carbonyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]-4-amino-4-oxobutanoic acid Chemical compound CC[C@H](C)[C@@H]1NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]2CCCN2C(=O)[C@@H](NC(=O)CNC(=O)[C@@H]2CCCN2C(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]2CSSC[C@H](NC1=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]3CCCN3C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC1=O)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)O)[C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N2)[C@@H](C)O PKTAYNJCGHSPDR-JNYFXXDFSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- LQNAGRRRLFOXGG-UHFFFAOYSA-N 3-(oxiran-2-yl)prop-2-enamide Chemical compound NC(=O)C=CC1CO1 LQNAGRRRLFOXGG-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102220546611 Albumin_H91A_mutation Human genes 0.000 description 1
- 102220495837 Alkaline ceramidase 1_Y49A_mutation Human genes 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101710192393 Attachment protein G3P Proteins 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 101800005049 Beta-endorphin Proteins 0.000 description 1
- 101500028443 Bos taurus Growth hormone-binding protein Proteins 0.000 description 1
- 101000583086 Bunodosoma granuliferum Delta-actitoxin-Bgr2b Proteins 0.000 description 1
- 101100075830 Caenorhabditis elegans mab-5 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102220614803 Calmodulin-3_Y92A_mutation Human genes 0.000 description 1
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 1
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 1
- 102100021809 Chorionic somatomammotropin hormone 1 Human genes 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 101710136772 Crambin Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 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
- 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
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 101100176785 Drosophila melanogaster gskt gene Proteins 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010049140 Endorphins Proteins 0.000 description 1
- 102000009025 Endorphins Human genes 0.000 description 1
- 241000702371 Enterobacteria phage f1 Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 101000867232 Escherichia coli Heat-stable enterotoxin II 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
- 108010060374 FSH Receptors Proteins 0.000 description 1
- 102000008175 FSH Receptors Human genes 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101710112780 Gene 1 protein Proteins 0.000 description 1
- 101710122194 Gene 2 protein Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WZSHYFGOLPXPLL-RYUDHWBXSA-N Gly-Phe-Glu Chemical compound NCC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(O)=O)C(O)=O WZSHYFGOLPXPLL-RYUDHWBXSA-N 0.000 description 1
- LBDXVCBAJJNJNN-WHFBIAKZSA-N Gly-Ser-Cys Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O LBDXVCBAJJNJNN-WHFBIAKZSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- OLIFSFOFKGKIRH-WUJLRWPWSA-N Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CN OLIFSFOFKGKIRH-WUJLRWPWSA-N 0.000 description 1
- 206010056438 Growth hormone deficiency Diseases 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
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 description 1
- 108091029795 Intergenic region 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
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- YRNRVKTYDSLKMD-KKUMJFAQSA-N Lys-Ser-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YRNRVKTYDSLKMD-KKUMJFAQSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 101800001751 Melanocyte-stimulating hormone alpha Proteins 0.000 description 1
- 101800000992 Melanocyte-stimulating hormone beta Proteins 0.000 description 1
- 108010086093 Mung Bean Nuclease Proteins 0.000 description 1
- 102220478107 Myc box-dependent-interacting protein 1_H64A_mutation Human genes 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 101710149086 Nuclease S1 Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108700006385 OmpF Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102220505611 Phospholipase A and acyltransferase 4_H21L_mutation Human genes 0.000 description 1
- 108010003044 Placental Lactogen Proteins 0.000 description 1
- 239000000381 Placental Lactogen Substances 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 102220472514 Protein ENL_H18R_mutation Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 102000028391 RNA cap binding Human genes 0.000 description 1
- 108091000106 RNA cap binding Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 101710137426 Replication-associated protein G2P Proteins 0.000 description 1
- 235000013290 Sagittaria latifolia Nutrition 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 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 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- QPMSXSBEVQLBIL-CZRHPSIPSA-N ac1mix0p Chemical compound C1=CC=C2N(C[C@H](C)CN(C)C)C3=CC(OC)=CC=C3SC2=C1.O([C@H]1[C@]2(OC)C=CC34C[C@@H]2[C@](C)(O)CCC)C2=C5[C@]41CCN(C)[C@@H]3CC5=CC=C2O QPMSXSBEVQLBIL-CZRHPSIPSA-N 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 229920006243 acrylic copolymer Polymers 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000012223 aqueous fraction 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
- 238000000149 argon plasma sintering Methods 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
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 230000001746 atrial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000037429 base substitution 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
- 108010042362 beta-Lipotropin Proteins 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- VDQQXEISLMTGAB-UHFFFAOYSA-N chloramine T Chemical compound [Na+].CC1=CC=C(S(=O)(=O)[N-]Cl)C=C1 VDQQXEISLMTGAB-UHFFFAOYSA-N 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 235000015246 common arrowhead Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 125000000731 cysteino group Chemical group [H]SC([H])([H])C([H])(C(O[H])=O)N([H])[*] 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 108010075816 gamma-Lipotropin Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 108010067006 heat stable toxin (E coli) Proteins 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 239000000960 hypophysis hormone Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000013383 initial experiment Methods 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 101150109249 lacI gene Proteins 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000000466 oxiranyl group Chemical group 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 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
- 239000002244 precipitate Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 102200095125 rs104893679 Human genes 0.000 description 1
- 102200082878 rs33935445 Human genes 0.000 description 1
- 102220151657 rs763546006 Human genes 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003093 somatogenic effect Effects 0.000 description 1
- 108010033419 somatotropin-binding protein Proteins 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960000874 thyrotropin Drugs 0.000 description 1
- 230000001748 thyrotropin Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960001479 tosylchloramide sodium Drugs 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- YNDXUCZADRHECN-JNQJZLCISA-N triamcinolone acetonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O YNDXUCZADRHECN-JNQJZLCISA-N 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 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 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 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
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/14011—Details ssDNA Bacteriophages
- C12N2795/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/14011—Details ssDNA Bacteriophages
- C12N2795/14111—Inoviridae
- C12N2795/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- G01N2333/575—Hormones
- G01N2333/61—Growth hormones [GH] (Somatotropin)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/802—Protein-bacteriophage conjugates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S930/00—Peptide or protein sequence
- Y10S930/01—Peptide or protein sequence
- Y10S930/12—Growth hormone, growth factor other than t-cell or b-cell growth factor, and growth hormone releasing factor; related peptides
Definitions
- This invention relates to the preparation and systematic selection of novel binding proteins having altered binding properties for a target molecule. Specifically, this invention relates to methods for producing foreign polypeptides mimicking the binding activity of naturally occurring binding partners. In preferred embodiments, the invention is directed to the preparation of therapeutic or diagnostic compounds that mimic proteins or nonpeptidyl molecules such a hormones, drugs and other small molecules, particularly biologically active molecules such as growth hormone.
- Binding partners are substances that specifically bind to one another, usually through noncovalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
- Binding partners have been produced in the past by a variety of methods including; harvesting them from nature (e.g., antibody-antigen, and ligand-receptor pairings) and by adventitious identification (e.g. traditional drug development employing random screening of candidate molecules). In some instances these two approaches have been combined. For example, variants of proteins or polypeptides, such as polypeptide fragments, have been made that contain key functional residues that participate in binding. These polypeptide fragments, in turn, have been derivatized by methods akin to traditional drug development. An example of such derivitization would include strategies such as cyclization to conformationally constrain a polypeptide fragment to produce a novel candidate binding partner.
- Geysen In an attempt to overcome these problems, Geysen (Geysen, Immun. Today. 6:364-369 [1985]); and (Geysen et al., Mol. Immun., 23:709-715 [1986]) has proposed the use of polypeptide synthesis to provide a framework for systematic iterative binding partner identification and preparation.
- short polypeptides such as dipeptides
- the most active dipeptides are then selected for an additional round of testing comprising linking, to the starting dipeptide, an additional residue (or by internally modifying the components of the original starting dipeptide) and then screening this set of candidates for the desired activity. This process is reiterated until the binding partner having the desired properties is identified.
- the Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure.
- random interactions accelerate among the various substituent groups of the polypeptide so that a true random population of interactive molecules having reproducible higher order structure becomes less and less attainable.
- interactions between side chains of amino acids, which are sequentially widely separated but which are spatially neighbors freely occur.
- sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule.
- Such complex interactions are facilitated by the flexibility of the polyamide backbone of the polypeptide candidates.
- candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.
- a final problem with the iterative polypeptide method of Geysen is that, at present, there are no practical methods with which a great diversity of different peptides can be produced, screened and analyzed.
- the total number of all combinations of hexapeptides that must be synthesized is 64,000,000.
- Even having prepared such a diversity of peptides there are no methods available with which mixtures of such a diversity of peptides can be rapidly screened to select those peptides having a high affinity for the target molecule.
- each “adherent” peptide must be recovered in amounts large enough to carry out protein sequencing.
- fusion phage have been shown to be useful for displaying short mutated peptide sequences for identifying peptides that may react with antibodies (Scott et al., Science 249: 386-390, [1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A 87: 6378-6382, [1990]) or a foreign protein (Devlin et al, Science, 249: 404-406 [1990]).
- fusion phage there are, however, several important limitations in using such “fusion phage” to identify altered peptides or proteins with new or enhanced binding properties.
- prior art methods have been unable to select peptides from a library having the highest binding affinity for a target molecule.
- Ladner WO 90/02802 discloses a method for selecting novel binding proteins displayed on the outer surface of cells and viral particles where it is contemplated that the heterologous proteins may have up to 164 amino acid residues. The method contemplates isolating and amplifying the displayed proteins to engineer a new family of binding proteins having desired affinity for a target molecule. More specifically, Ladner discloses a “fusion phage” displaying proteins having “initial protein binding domains” ranging from 46 residues (crambin) to 164 residues (T4 lysozyme) fused to the M13 gene III coat protein.
- Ladner teaches the use of proteins “no larger than necessary” because it is easier to arrange restriction sites in smaller amino acid sequences and prefers the 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI).
- Small fusion proteins such as BPTI
- BPTI small fusion proteins
- T4 lysozyme small target molecules
- the preferred protein, BPTI is proposed to be fused to gene III at the site disclosed by Smith et al. or de la Cruz et al, J. Biol.
- hGH Human growth hormone
- hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al, (1986) Endocrine Reviews 7, 169). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W., et al, [1987 ] Nature 330, 537) or prolactin receptor (Boutin, J. M., et al, [1988 ] Ce: 53, 69). The cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, C.
- hGH Human growth hormone
- It is another object of this invention to prepare candidate binding substances comprising fusion proteins of a phage coat protein and a heterologous polypeptide where the polypeptide is greater than 100 amino acids in length and may be more than one subunit and is displayed on a phagemid particle where the polypeptide is encoded by the phagemid genome.
- Still another object of the invention is the production of growth hormone variants that exhibit stronger affinity for growth hormone receptor and binding protein.
- a method for selecting novel binding polypeptides comprising: (a) constructing a replicable expression vector comprising a first gene encoding a polypeptide, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcription regulatory element operably linked to the first and second genes, thereby forming a gene fusion encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that
- the method for selecting novel binding proteins where the proteins are composed of more than one subunit is achieved by selecting novel binding peptides comprising constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein; mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors; transforming suitable host cells with the vectors; infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface
- the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 1%. Also preferably, amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably the amount is less than 20%.
- the expression vector will further contain a secretory signal sequences fused to the DNA encoding each subunit of the polypeptide, and the transcription regulatory element will be a promoter system.
- Preferred promoter systems are selected from; Lac Z, ⁇ PL , TAC, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
- the first gene will encode a mammalian protein, preferably the protein will be selected from; human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide,
- the first gene will encode a polypeptide of one or more subunits containing more than about 100 amino acid residues and will be folded to form a plurality of rigid secondary structures displaying a plurality of amino acids capable of interacting with the target.
- the first gene will be mutated at codons corresponding to only the amino acids capable of interacting with the target so that the integrity of the rigid secondary structures will be preserved.
- helper phage selected from; M13KO7, M13R408, M13-VCS, and Phi X 174.
- the preferred helper phage is M13KO7
- the preferred coat protein is the M13 Phage gene III coat protein.
- the preferred host is E. coli , and protease deficient strains of E. coli . Novel hGH variants selected by the method of the present invention have been detected.
- Phagemid expression vectors were constructed that contain a suppressible termination codon functionally located between the nucleic acids encoding the polypeptide and the phage coat protein.
- FIG. 1 Strategy for displaying large proteins on the surface of filamentous phage and enriching for altered receptor binding properties.
- a plasmid, phGH-M13gIII was constructed that fuses the entire coding sequence of hGH to the carboxyl terminal domain of M13 gene III. Transcription of the fusion protein is under control of the lac promoter/operator sequence, and secretion is directed by the stII signal sequence.
- Phagemid particles are produced by infection with the “helper” phage, M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor.
- the wild-type gene III (derived from the M13KO7 phage) is diagramed by 4-5 copies of the multiple arrows on the tip of the phage, and the fusion protein (derived from the phagemid, phGH-M13gIII) is indicated schematically by the folding diagram of hGH replacing the arrow head.
- FIG. 2 Immunoblot of whole phage particles shows that hGH comigrates with phage.
- Phagemid particles purified in a cesium chloride gradient were loaded into duplicate wells and electrophoresed through a 1% agarose gel in 375 mM Tris, 40 mM glycine pH 9.6 buffer. The gel was soaked in transfer buffer (25 mM Tris, pH 8.3, 200 mM glycine, 20% methanol) containing 2% SDS and 2% ⁇ -mercaptoethanol for 2 hours, then rinsed in transfer buffer for 6 hours. The proteins in the gel were then electroblotted onto immobilon membranes (Millipore).
- the membrane containing one set of samples was stained with Coomassie blue to show the position of the phage proteins (A).
- the duplicate membrane was immuno-stained for hGH by reacting the membrane with polyclonal rabbit anti-hGH antibodies followed by reaction with horseradish peroxidase conjugated goat anti-rabbit IgG antibodies (B).
- Lane 1 contains the M13KO7 parent phage and is visible only in the Coomassie blue stained membrane, since it lacks hGH.
- Lanes 2 and 3 contain separate preparations of the hormone phagemid particles which is visible both by Coomassie and hGH immuno-staining.
- the difference in migration distance between the parent M13KO7 phage and hormone phagemid particles reflects the different size genomes that are packaged within (8.7 kb vs. 5.1 kb, respectively).
- FIG. 3 Summary diagram of steps in the selection process for an hGH-phage library randomized at codons 172, 174, 176, and 178.
- the template molecules, pH0415, containing a unique KpnI restriction site and the hGH(R178G,I179T) gene was mutagenized as described in the text and electrotransformed into E. coli strain WJM101 to obtain the initial phagemid library, Library 1.
- An aliquot (approximately 2%) from Library 1 was used directly in an initial selection round as described in the text to yield Library 1G.
- dsDNA double-stranded DNA (dsDNA) was prepared from Library I, digested with restriction enzyme KpnI to eliminate template background, and electrotransformed into WJM101 to yield Library 2.
- FIG. 4 Structural model of hGH derived from a 2.8 ⁇ folding diagram of porcine growth hormone determined crystallographically. Location of residues in hGH that strongly modulate its binding to the hGH-binding protein are within the shaded circle. Alanine substitutions that cause a greater than tenfold reduction ( ⁇ ), a four- to tenfold reduction ( ⁇ ), or increase ( ⁇ ), or a two- to fourfold reduction ( ⁇ ), in binding affinity are indicated.
- Helical wheel projections in the regions of ⁇ -helix reveal their amphipathic quality. Blackened, shaded, or nonshaded residues are charged, polar, or nonpolar, respectively. In helix-4 the most important residues for mutation are on the hydrophilic face.
- FIG. 5 Amino acid substitutions at positions 172, 174, 176 and 178 of hGH (The notation, e.g. KSYR, denotes hGH mutant 172K/174S/176Y/178R) found after sequencing a number of clones from rounds 1 and 3 of the selection process for the pathways indicated (hGH elution; Glycine elution; or Glycine elution after pre-adsorption).
- Non-functional sequences i.e. vector background, or other prematurely terminated and/or frame-shifted mutants
- Functional sequences which contained a non-silent, spurious mutation i.e. outside the set of target residues
- Protein sequences which appeared more than once among all the sequenced clones, but with different DNA sequences, are marked with a “#”. Protein sequences which appeared more than once among the sequenced clones and with the same DNA sequence are marked with a “*”. Note that after three rounds of selection, 2 different contaminating sequences were found; these clones did not correspond to cassette mutants, but to previously constructed hormone phage.
- the pS0643 contaminant corresponds to wild-type hGH-phage (hGH “KEFR” (SEQ ID NO:44)).
- the pH0457 contaminant which dominates the third-round glycine-selected pool of phage, corresponds to a previously identified mutant of hGH, “KSYR.”
- K a previously identified mutant of hGH
- the amplification of these contaminants emphasizes the ability of the hormone-phage selection process to select for rarely occurring mutants.
- the convergence of sequences is also striking in all three pathways: R or K occurs most often at positions 172 and 178; Y or F occurs most often at position 176; and S, T, A, and other residues occur at position 174.
- FIG. 6 Sequences from phage selected on hPRLbp-beads in the presence of zinc. The notation is as described in FIG. 5 . Here, the convergence of sequences is not predictable, but there appears to be a bias towards hydrophobic sequences under the most stringent (Glycine) selection conditions; L W and P residues are frequently found in this pool.
- FIG. 7 Sequences from phage selected on hPRLbp-beads in the absence of zinc. The notation is as described in FIG. 5 . In contrast to the sequences of FIG. 6 , these sequences appear more hydrophilic. After 4 rounds of selection using hGH elution, two clones (ANHQ (SEQ ID NO:45), and TLDT/171V (SEQ ID NO:108)) dominate the pool.
- FIG. 8 Sequences from phage selected on blank beads. The notation is as described in FIG. 5 . After three rounds of selection with glycine elution, no siblings were observed and a background level of non-functional sequences remained.
- FIG. 9 Construction of phagemid fl ori from pHO415.
- This vector for cassette mutagenesis and expression of the hGH-gene III fusion protein was constructed as follows. Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter.
- Mutagenesis was carried out with the oligonucleotide 5′-GGC-AGC-TGT-GGC-T TC-TAG-A GT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduced a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH.
- FIG. 10 A. Diagram of plasmid pDH188 insert containing the DNA encoding the light chain and heavy chain (variable and constant domain 1) of the F ab humanized antibody directed to the HER-2 receptor.
- V L and V H are the variable regions for the light and heavy chains, respectively.
- C k is the constant region of the human kappa light chain.
- CH1 G1 is the first constant region of the human gamma 1 chain. Both coding regions start with the bacterial st II signal sequence.
- B A schematic diagram of the entire plasma pDH188 containing the insert described in 5 A. After transformation of the plasmid into E. coli SR101 cells and the addition of helper phage, the plasmid is packaged into phage particles. Some of these particles display the F ab -p III fusion (where p III is the protein encoded by the M13 gene III DNA). The segments in the plasmid figure correspond to the insert shown in 5 A.
- FIG. 11A through C are collectively referred to here as FIG. 11 .
- the amino acid sequence of the light chain is also shown (Seq. ID No:25), as is the amino acid sequence of the heavy chain p III fusion (Seq. ID No:26).
- FIG. 12 Enrichment of wild-type 4D5 F ab phagemid from variant F ab phagemid.
- Mixtures of wild-type phagemid and variant 4D5 F ab phagemid in a ratio of 1:1,000 were selected on plates coated with the extra-cellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid were infected into E. coli and plasmid DNA was prepared. This plasmid DNA was then digested with Eco RV and Pst I, separated on a 5% polyacrylamide gel, and stained with ethidium bromide. The bands were visualized under UV light. The bands due to the wild-type and variant plasmids are marked with arrows.
- the first round of selection was eluted only under acid conditions; subsequent rounds were eluted with either an acid elution (left side of Figure) or with a humanized 4D5 antibody wash step prior to acid elution (right side of Figure) using methods described in Example VIII.
- H91A amino acid histidine at position 91 on the V L chain mutated to alanine; indicated as ‘A’ lanes in Figure
- Y49A amino acid tyrosine at position 49 on the V L chain mutated to alanine; indicated as ‘B’ lanes in the Figure
- Y92A amino acid tyrosine at position 92 on the V L chain mutated to alanine; indicated as ‘C’ lanes in the Figure.
- Amino acid position numbering is according to Kabat et al., ( Sequences of proteins of immunological interest, 4th ed., U.S. Dept of Health and Human Services, Public Health Service, Nat'l. Institute of Health, Bethesda, Md. [1987]).
- FIG. 13 The Scatchard analysis of the RIA affinity determination described in Experimental Protocols is shown here.
- the amount of labeled ECD antigen that is bound is shown on the x-axis while the amount that is bound divided by the amount that is free is shown on the y-axis.
- the slope of the line indicates the K a ; the calculated K d is 1/K a .
- the method of the instant invention comprises a method for selecting novel binding polypeptides, such as protein ligands, having a desired, usually high, affinity for a target molecule from a library of structurally related binding polypeptides.
- the library of structurally related polypeptides, fused to a phage coat protein, is produced by mutagenesis and, preferably, a single copy of each related polypeptide is displayed on the surface of a phagemid particle containing DNA encoding that polypeptide.
- These phagemid particles are then contacted with a target molecule and those particles having the highest affinity for the target are separated from those of lower affinity.
- the high affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained.
- novel binding polypeptides or ligands produced by the method of this invention are useful per se as diagnostics or therapeutics (eg. agonists or antagonists) used in treatment of biological organisms. Structural analysis of the selected polypeptides may also be used to facilitate rational drug design.
- binding polypeptide as used herein is meant any polypeptide that binds with a selectable affinity to a target molecule.
- the polypeptide will be a protein that most preferably contains more than about 100 amino acid residues.
- the polypeptide will be a hormone or an antibody or a fragment thereof.
- high affinity as used herein is meant an affinity constant (K d ) of ⁇ 10 ⁇ 5 M and preferably ⁇ 10 ⁇ 7 M under physiological conditions.
- target molecule as used herein is meant any molecule, not necessarily a protein, for which it is desirable to produce a ligand.
- the target will be a protein and most preferably the target will be a receptor, such as a hormone receptor.
- humanized antibody as used herein is meant an antibody in which the complementarity-determining regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework.
- human antibody framework is meant the entire human antibody excluding the CDRs.
- the first step in the method of this invention is to choose a polypeptide having rigid secondary structure exposed to the surface of the polypeptide for display on the surface of a phage.
- polypeptide as used herein is meant any molecule whose expression can be directed by a specific DNA sequence.
- the polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
- rigid secondary structure any polypeptide segment exhibiting a regular repeated structure such as is found in; ⁇ -helices, 3 10 helices, ⁇ -helices, parallel and antiparallel ⁇ -sheets, and reverse turns.
- Certain “non-ordered” structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or “patch” of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns.
- the geometry of these rigid secondary structures is well defined by ⁇ and ⁇ torsional angles about the ⁇ -carbons of the peptide “backbone”.
- the requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or “patch” of amino acid residues that can be exposed to and bind with a target molecule. It is primarily these amino acid residues that are replaced by mutagenesis that form the “library” of structurally related (mutant) binding polypeptides that are displayed on the surface of the phage and from which novel polypeptide ligands are selected. Mutagenesis or replacement of amino acid residues directed toward the interior of the polypeptide is generally avoided so that the overall structure of the rigid secondary structure is preserved. Some replacement of amino acids on the interior region of the rigid secondary structures, especially with hydrophobic amino acid residues, may be tolerated since these conservative substitutions are unlikely to distort the overall structure of the polypeptide.
- hGH amino acids 167, 171, 175 and 179 were phagemid selected.
- hGH amino acids 10, 14, 18 and 21 were phagemid selected.
- Optimum amino acid changes from a previous cycle may be incorporated into the polypeptide before the next cycle of selection. For example, hGH amino acids substitution 174 (serine) and 176 (tyrosine) were incorporated into the hGH before the phagemid selection of hGH amino acids 167, 171, 175 and 179.
- the amino acid residues that form the binding domain of the polypeptide will not be sequentially linked and may reside on different subunits of the polypeptide. That is, the binding domain tracks with the particular secondary structure at the binding site and not the primary structure.
- mutations will be introduced into codons encoding amino acids within a particular secondary structure at sites directed away from the interior of the polypeptide so that they will have the potential to interact with the target.
- FIG. 2 shows the location of residues in hGH that are known to strongly modulate its binding to the hGH-binding protein (Cunningham et al., Science 247:1461-1465 [1990]).
- representative sites suitable for mutagenesis would include residues 172, 174, 176, and 178 on helix-4, as well as residue 64 located in a “non-ordered” secondary structure.
- polypeptide chosen as a ligand to a target normally bind to that target.
- a glycoprotein hormone such as TSH can be chosen as a ligand for the FSH receptor and a library of mutant TSH molecules are employed in the method of this invention to produce novel drug candidates.
- polypeptides that binds to a target molecule, and includes antibodies.
- Preferred polypeptides are those that have pharmaceutical utility. More preferred polypeptides include; a growth hormone, including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroid stimulating hormone; thyroxine; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; leutinizing hormone; glucagon; factor VIII; an antibody; lung surfactant; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator (t-PA); bombesin; factor IX, thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prore
- polypeptides of this invention are human growth hormone, and atrial naturetic peptides A, B, and C, endotoxin, subtilisin, trypsin and other serine proteases.
- polypeptide hormones that can be defined as any amino acid sequence produced in a first cell that binds specifically to a receptor on the same cell type (autocrine hormones) or a second cell type (non-autocrine) and causes a physiological response characteristic of the receptor-bearing cell.
- polypeptide hormones include cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, ⁇ or ⁇ -melanocyte-stimulating hormone, ⁇ -lipotropin, ⁇ -lipotropin and the endorphins; hypothalmic release-inhibiting hormones such as corticotropin-release factor, growth hormone release-inhibiting hormone, growth hormone-release factor; and other polypeptide hormones such as atrial natriuretic peptides A, B or C.
- cytokines such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, ⁇ or ⁇ -mel
- the gene encoding the desired polypeptide i.e., a polypeptide with a rigid secondary structure
- the DNA encoding the gene may be chemically synthesized (Merrfield, J. Am. Chem. Soc., 85 :2149 [1963]).
- the sequence of the gene is not known, or if the gene has not previously been isolated, it may be cloned from a cDNA library (made from RNA obtained from a suitable tissue in which the desired gene is expressed) or from a suitable genomic DNA library.
- probes include monoclonal or polyclonal antibodies (provided that the cDNA library is an expression library), oligonucleotides, and complementary or homologous cDNAs or fragments thereof.
- the probes that may be used to isolate the gene of interest from genomic DNA libraries include cDNAs or fragments thereof that encode the same or a similar gene, homologous genomic DNAs or DNA fragments, and oligonucleotides. Screening the cDNA or genomic library with the selected probe is conducted using standard procedures as described in chapters 10-12 of Sambrook et al., supra.
- PCR polymerase chain reaction methodology
- the gene may be inserted into a suitable vector (preferably a plasmid) for amplification, as described generally in Sambrook et al., supra.
- a suitable vector preferably a plasmid
- Plasmid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Plasmid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
- Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter, the bacteriophage ⁇ PL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter.
- the lac Z promoter system the alkaline phosphatase pho A promoter
- the bacteriophage ⁇ PL promoter a temperature sensitive promoter
- the tac promoter a hybrid trp-lac promoter that is regulated by the lac repressor
- tryptophan promoter a hybrid trp-lac promoter that is regulated by the lac repressor
- the tryptophan promoter a hybrid trp-lac promoter that is regulated by the lac repressor
- the tryptophan promoter the bacteriophage T7 promoter.
- Preferred promoters for practicing this invention are those that can be tightly regulated such that expression of the fusion gene can be controlled. It is believed that the problem that went unrecognized in the prior art was that display of multiple copies of the fusion protein on the surface of the phagemid particle lead to multipoint attachment of the phagemid with the target. It is believed this effect, referred to as the “chelate effect”, results in selection of false “high affinity” polypeptides when multiple copies of the fusion protein are displayed on the phagemid particle in close proximity to one another so that the target was “chelated”. When multipoint attachment occurs, the effective or apparent Kd may be as high as the product of the individual Kds for each copy of the displayed fusion protein. This effect may be the reason Cwirla and coworkers supra were unable to separate moderate affinity peptides from higher affinity peptides.
- Preferred promoters used to practice this invention are the lac Z promoter and the pho A promoter.
- the lac Z promoter is regulated by the lac repressor protein lac i, and thus transcription of the fusion gene can be controlled by manipulation of the level of the lac repressor protein.
- the phagemid containing the lac Z promotor is grown in a cell strain that contains a copy of the lac i repressor gene, a repressor for the lac Z promotor.
- Exemplary cell strains containing the lac i gene include JM 101 and XL1-blue.
- the host cell can be cotransfected with a plasmid containing both the repressor lac i and the Lac Z promotor.
- phagmide particles containing the Lac Z promoter are grown in cell strains containing the lac i gene and the cell strains are cotransfected with a plasmid containing both the lac Z and lac i genes.
- an inducer such as isopropylthiogalactoside (IPTG).
- IPTG isopropylthiogalactoside
- the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles).
- the most preferred promoter used to practice this invention is pho A. This promoter is believed to be regulated by the level of inorganic phosphate in the cell where the phosphate acts to down-regulate the activity of the promoter. Thus, by depleting cells of phosphate, the activity of the promoter can be increased. The desired result is achieved by growing cells in a phosphate enriched medium such as 2YT or LB thereby controlling the expression of the gene III fusion.
- One other useful component of vectors used to practice this invention is a signal sequence.
- This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell.
- the DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence.
- Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:193 [1983]), MalE, PhoA and other genes.
- a preferred prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence as described by Chang et al., Gene, 55: 189 [1987].
- STII enterotoxin II
- phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell.
- amp ampicillin resistance gene
- tet tetracycline resistance gene
- Suitable vectors comprising the aforementioned components as well as the gene encoding the desired polypeptide (gene 1) are prepared using standard recombinant DNA procedures as described in Sambrook et al. supra. Isolated DNA fragments to be combined to form the vector are cleaved, tailored, and ligated together in a specific order and orientation to generate the desired vector.
- the DNA is cleaved using the appropriate restriction enzyme or enzymes in a suitable buffer.
- a suitable buffer In general, about 0.2-1 ⁇ g of plasmid or DNA fragments is used with about 1-2 units of the appropriate restriction enzyme in about 20 ⁇ l of buffer solution.
- Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the manufacturers of the restriction enzymes. Generally, incubation times of about one or two hours at 37° C. are adequate, although several enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and the DNA is recovered from the aqueous fraction by precipitation with ethanol.
- the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the sticky ends commonly produced by endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C. with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
- the cleaved DNA fragments may be size-separated and selected using DNA gel electrophoresis.
- the DNA may be electrophoresed through either an agarose or a polyacrylamide matrix. The selection of the matrix will depend on the size of the DNA fragments to be separated.
- the DNA is extracted from the matrix by electroelution, or, if low-melting agarose has been used as the matrix, by melting the agarose and extracting the DNA from it, as described in sections 6.30-6.33 of Sambrook et al., supra.
- the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
- the solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
- the vector is at first linearized by cutting with the appropriate restriction endonuclease(s).
- the linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. The phosphatasing prevents self-ligation of the vector during the ligation step.
- Prokaryotes are the preferred host cells for this invention.
- Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCC number 31,537), E. coli XL-1 Blue (stratagene), and E. coli B; however many other strains of E. coli , such as HB101, NM522, NM538, NM539, and many other species and genera of prokaryotes may be used as well.
- E. coli strain JM101 E. coli K12 strain 294
- E. coli strain W3110 ATCC number 27,325
- E. coli X1776 ATCC number 31,537
- E. coli XL-1 Blue stratagene
- E. coli B E. coli B
- many other strains of E. coli such as HB101, NM522, NM53
- bacilli such as Bacillus subtilis
- enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans
- various Pseudomonas species may all be used as hosts.
- Transformation of prokaryotic cells is readily accomplished using the calcium chloride method as described in section 1.82 of Sambrook et al., supra.
- electroporation may be used to transform these cells.
- the transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
- tet tetracycline
- amp amp
- Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale preparation of DNA and the large-scale preparation of DNA as described in sections 1.25-1.33 of Sambrook et al., supra. The isolated DNA can be purified by methods known in the art such as that described in section 1.40 of Sambrook et al., supra. This purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is generally performed by either the method of Messing et al. Nucleic Acids Res., 9:309 [1981] or by the method of Maxam et al. Meth. Enzymol., 65: 499 [1980].
- This invention contemplates fusing the gene enclosing the desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is generated during transcription.
- Gene 2 is typically a coat protein gene of a phage, and preferably it is the phage M13 gene III coat protein, or a fragment thereof. Fusion of genes 1 and 2 may be accomplished by inserting gene 2 into a particular site on a plasmid that contains gene 1, or by inserting gene 1 into a particular site on a plasmid that contains gene 2.
- Insertion of a gene into a plasmid requires that the plasmid be cut at the precise location that the gene is to be inserted. Thus, there must be a restriction endonuclease site at this location (preferably a unique site such that the plasmid will only be cut at a single location during restriction endonuclease digestion).
- the plasmid is digested, phosphatased, and purified as described above.
- the gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be accomplished if the ends of the plasmid are compatible with the ends of the gene to be inserted.
- the DNAs can be ligated together directly using a ligase such as bacteriophage T4 DNA ligase and incubating the mixture at 16° C. for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of Sambrook et al., supra. If the ends are not compatible, they must first be made blunt by using the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both of which require the four deoxyribonucleotide triphosphates to fill-in overhanging single-stranded ends of the digested DNA.
- a ligase such as bacteriophage T4 DNA ligase
- the ends may be blunted using a nuclease such as nuclease S1 or mung-bean nuclease, both of which function by cutting back the overhanging single strands of DNA.
- the DNA is then religated using a ligase as described above.
- oligonucleotide linkers may be used. The linkers serve as a bridge to connect the plasmid to the gene to be inserted. These linkers can be made synthetically as double stranded or single stranded DNA using standard methods.
- the linkers have one end that is compatible with the ends of the gene to be inserted; the linkers are first ligated to this gene using ligation methods described above.
- the other end of the linkers is designed to be compatible with the plasmid for ligation.
- care must be taken to not destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid.
- it may be necessary to design the linkers such that they code for part of an amino acid, or such that they code for one or more amino acids.
- termination codons are UAG (amber), UM (ocher) and UGA (opel). (Microbiology, Davis et al. Harper & Row, New York, 1980, pages 237, 245-47 and 274).
- the termination codon expressed in a wild type host cell results in the synthesis of the gene 1 protein product without the gene 2 protein attached.
- growth in a suppressor host cell results in the synthesis of detectable quantities of fused protein.
- Such suppressor host cells contain a tRNA modified to insert an amino acid in the termination codon position of the mRNA thereby resulting in production of detectible amounts of the fusion protein.
- suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al., BioTechniques 5, 376-379 [1987]). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide.
- the suppressible codon may be inserted between the first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein.
- the suppressible termination codon may be inserted adjacent to the fusion site by replacing the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein.
- the polypeptide When the phagemid is grown in a non-suppressor host cell, the polypeptide is synthesized substantially without fusion to the phage coat protein due to termination at the inserted suppressible triplet encoding UAG, UAA, or UGA. In the non-suppressor cell the polypeptide is synthesized and secreted from the host cell due to the absence of the fused phage coat protein which otherwise anchored it to the host cell.
- Gene 1 encoding the desired polypeptide may be altered at one or more selected codons.
- An alteration is defined as a substitution, deletion, or insertion of one or more codons in the gene encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the unaltered or native sequence of the same polypeptide.
- the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule.
- the alterations may be produced be a variety of methods known in the art. These methods include but are not limited to oligonucleotide-mediated mutagenesis and cassette mutagenesis.
- Oligonucleotide-mediated mutagenesis is preferred method for preparing substitution, deletion, and insertion variants of gene 1. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 [1987]. Briefly, gene 1 is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of the plasmid containing the unaltered or native DNA sequence of gene 1. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template will thus incorporate the oligonucleotide primer, and will code for the selected alteration in gene 1.
- oligonucleotides of at least 25 nucleotides in length are used.
- An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
- the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. Proc. Nat'l. Acad. Sci. USA, 75: 5765 [1978].
- the DNA template can only be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13 mp18 and M13 mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. Meth. Enzymol., 153: 3 [1987]. Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.
- the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions.
- a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
- a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of gene 1, and the other strand (the original template) encodes the native, unaltered sequence of gene 1.
- This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. Coli JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.
- the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s).
- the modifications are as follows:
- the single-stranded oligonucleotide is annealed to the single-stranded template as described above.
- a mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP) is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex.
- this new strand of DNA Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated.
- this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion.
- the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded.
- a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase.
- This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.
- Mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
- a separate oligonucleotide is generated for each amino acid to be substituted.
- the oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.
- the alternative method involves two or more rounds of mutagenesis to produce the desired mutant.
- the first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated.
- the second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template.
- this template already contains one or more mutations.
- the oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis.
- This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
- This method is also a preferred method for preparing substitution, deletion, and insertion variants of gene 1.
- the method is based on that described by Wells et al. Gene, 34:315 [1985].
- the starting material is the plasmid (or other vector) comprising gene 1, the gene to be mutated.
- the codon(s) in gene 1 to be mutated are identified.
- a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques.
- This double-stranded oligonucleotide is referred to as the cassette.
- This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
- This plasmid now contains the mutated DNA sequence of gene 1.
- this invention contemplates production of variants of a desired protein containing one or more subunits.
- Each subunit is typically encoded by separate gene.
- Each gene encoding each subunit can be obtained by methods known in the art (see, for example, Section II). In some instances, it may be necessary to obtain the gene encoding the various subunits using separate techniques selected from any of the methods described in Section II.
- all subunits can be regulated by the same promoter, typically located 5′ to the DNA encoding the subunits, or each may be regulated by separate promoter suitably oriented in the vector so that each promoter is operably linked to the DNA it is intended to regulate. Selection of promoters is carried out as described in Section III above.
- FIG. 10 In constructing a replicable expression vector containing DNA encoding the protein of interest having multiple subunits, the reader is referred to FIG. 10 where, by way of illustration, a vector is diagrammed showing DNA encoding each subunit of an antibody fragment.
- a vector is diagrammed showing DNA encoding each subunit of an antibody fragment.
- This figure shows that, generally, one of the subunits of the protein of interest will be fused to a phage coat protein such as M13 gene III. This gene fusion generally will contain its own signal sequence. A separate gene encodes the other subunit or subunits, and it is apparent that each subunit generally has its own signal sequence.
- FIG. 10 also shows that a single promoter can regulate the expression of both subunits. Alternatively, each subunit may be independently regulated by a different promoter.
- the protein of interest subunit-phage coat protein fusion construct can be made as described in Section IV above.
- DNA encoding each subunit in the vector may mutated in one or more positions in each subunit.
- preferred sites of mutagenesis correspond to codons encoding amino acid residues located in the complementarity-determining regions (CDR) of either the light chain, the heavy chain, or both chains.
- CDRs are commonly referred to as the hypervariable regions.
- Target proteins such as receptors
- glycoprotein hormone receptors may be prepared by the technique described by McFarland et al., Science 245:494-499 [1989]
- nonglycosylated forms expressed in E. coli are described by Fuh et al. J. Biol. Chem. 265:3111-3115 [1990]
- Other receptors can be prepared by standard methods.
- the purified target protein may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxylalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44 [1976], or by other means known in the art.
- the immobilized target is contacted with the library of phagemid particles under conditions suitable for binding of at least a portion of the phagemid particles with the immobilized target.
- the conditions including pH, ionic strength, temperature and the like will mimic physiological conditions.
- Binders having high affinity for the immobilized target are separated from those having a low affinity (and thus do not bind to the target) by washing. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
- Suitable host cells are infected with the binders and helper phage, and the host cells are cultured under conditions suitable for amplification of the phagemid particles. The phagemid particles are then collected and the selection process is repeated one or more times until binders having the desired affinity for the target molecule are selected.
- the library of phagemid particles may be sequentially contacted with more than one immobilized target to improve selectivity for a particular target.
- a ligand such as hGH has more than one natural receptor.
- both the growth hormone receptor and the prolactin receptor bind the hGH ligand. It may be desirable to improve the selectivity of hGH for the growth hormone receptor over the prolactin receptor. This can be achieved by first contacting the library of phagemid particles with immobilized prolactin receptor, eluting those with a low affinity (i.e.
- hGH mutant having a lower affinity for the prolactin receptor would have therapeutic utility even if the affinity for the growth hormone receptor were somewhat lower than that of wild type hGH.
- This same strategy may be employed to improve selectivity of a particular hormone or protein for its primary function receptor over its clearance receptor.
- an improved substrate amino acid sequence can be obtained. These may be useful for making better “cut sites” for protein linkers, or for better protease substrates/inhibitors.
- an immobilizable molecule e.g. hGH-receptor, biotin-avidin, or one capable of covalent linkage with a matrix
- the linker will preferably be from 3 to 10 amino acids in length and will act as a substrate for a protease.
- a phagemid will be constructed as described above where the DNA encoding the linker region is randomly mutated to produce a randomized library of phagemid particles with different amino acid sequences at the linking site.
- the library of phagemid particles are then immobilized on a matrix and exposed to a desired protease.
- Phagemid particles having preferred or better substrate amino acid sequences in the liner region for the desired protease will be eluted, first producing an enriched pool of phagemid particles encoding preferred linkers.
- These phagemid particles are then cycled several more times to produce an enriched pool of particles encoding consense sequence(s) (see examples XIII and XIV).
- the cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N>, et al., [1987 ] Gene 55 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979 ] Nature 281, 544; Gray et al., [1985 ] Gene 39, 247).
- the present invention describes novel hGH variants produced using the phagemid selection methods. Human growth hormone variants containing substitutions at positions 10, 14, 18, 21, 167, 171, 172, 174, 175, 176, 178 and 179 have been described. Those having higher binding affinities are described in Tables VII, XIII and XIV.
- Growth hormone variants may be administered and formulated in the same manner as regular growth hormone.
- the growth hormone variants of the present invention may be expressed in any-recombinant system which is capable of expressing native or met hGH.
- Therapeutic formulations of hGH for therapeutic administration are prepared for storage by mixing hGH having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers ( Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980)., 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; divalent metal ions such as zinc, cobalt or copper; 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
- Formulations of the present invention may additionally contain a pharmaceutically acceptable buffer, amino acid, bulking agent and/or non-ionic surfactant.
- a pharmaceutically acceptable buffer include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like; specific examples of these could include, trimethylamaine salts (“Tris buffer”), and disodium edetate.
- Tris buffer trimethylamaine salts
- the phagemids of the present invention may be used to produce quantities of the hGH variants free of the phage protein.
- pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- the hGH variant is secreted from the host and may be isolated from the culture medium.
- One or more of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I 179 may be replaced by any amino acid other than the one found in that position in naturally occurring hGH as indicated. Therefore, 1, 2, 3, 4, 5, 6, 7, or all 8 of the indicated amino acids, F10, M14, H18, H21, R167, D171, T175 and I 179, may be replaced by any of the other 19 amino acids out of the 20 amino acids listed below. In a preferred embodiment, all eight listed amino acids are replaced by another amino acid. The most preferred eight amino acids to be substituted are indicated in Table XIV in Example XII. Amino acid nomenclature.
- the plasmid phGH-M13gIII ( FIG. 1 ), was constructed from M13KO7 7 and the hGH producing plasmid, pBO473 (Cunningham, B. C., et al., Science, 243:1330-1336, [1989]).
- a synthetic oligonucleotide 5′-AGC-TGT-GGC-TTC- GGG-CCC -TTA-GCA-TTT-AAT-GCG-GTA-3′ (SEQ ID NO:2) was used to introduce a unique ApaI restriction site (underlined) into pBO473 after the final Phe191 codon of hGH.
- the oligonucleotide 5′-TTC-ACA-AAC-GAA- GGG-CCC -CTA-ATT-AAA-GCC-AGA-3′ was used to introduce a unique ApaI restriction site (underlined), and a Glu197-to-amber stop codon (hold lettering) into M13KO7 gene III.
- the oligonucleotide 5′-CAA-TAA-TAA-CGG- GCT-AGC -CAA-AAG-AAC-TGG-3′ introduces a unique NheI site (underlined) after the 3′ end of the gene III coding sequence.
- a 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and Cap binding site was produced by PCR of plasmid pUC119 using the oligonucleotides 5′-CACGACA GAATTC CCGACTGGAAA-3′ (SEQ ID NO:5) and 5′-CTGTT TCTAGA GTGAAATTGTTA-3′ (SEQ ID NO:6) that flank the desired lac sequences and introduce the EcoRI and XbaI restriction sites (underlined).
- This lac fragment was gel purified and ligated into the large EcoRI-XbaI fragment of pSO132 to create the plasmid, phGH-M13gIII.
- the sequences of all tailored DNA junctions were verified by the dideoxy sequence method (Sanger, F., et al. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, [1977]).
- the R64A variant hGH phagemid was constructed as follows: the NsiI-BglII mutated fragment of hGH (Cunningham et al. supra) encoding the Arg64 to Ala substitution (R64A) (Cunningham, B. C., Wells, J.
- Plasmids were transformed into a male strain of E. coli (JM101) and selected on carbenicillin plates. A single transformant was grown in 2 ml 2YT medium for 4 h at 37° C. and infected with 50 ⁇ l of M13KO7 helper phage. The infected culture was diluted into 30 ml 2YT, grown overnight, and phagemid particles were harvested by precipitation with polyethylene glycol (Vierra, J., Messing, J., Methods in Enzymology, 153:3-11, [1987]). Typical phagemid particle titers ranged from 2 to 5 ⁇ 10 11 cfu/ml. The particles were purified to homogeneity by CsCl density centrifugation (Day, L. A. J. Mol. Biol., 39:265-277, [1969]) to remove any fusion protein not attached to virions.
- Rabbit polyclonal antibodies to hGH were purified with protein A, and coated onto microtiter plates (Nunc) at a concentration of 2 ⁇ g/ml in 50 mM sodium carbonate buffer (pH 10) at 4° C. for 16-20 hours. After washing in PBS containing 0.05% Tween 20, hGH or hGH-phagemid particles were serially diluted from 2.0-0.002 nM in buffer A (50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg/ml bovine serum albumin, and 0.05% Tween 20). After 2 hours at room temperature (rt), the plates were washed well and the indicated Mab (Cunningham et al.
- Oxirane polyacrylamide beads (Sigma) were conjugated to the purified extracellular domain of the hGH receptor (hGHbp) (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 [1990]) containing an extra cysteine residue introduced by site-directed mutagenesis at position 237 that does not affect binding of hGH (J. Wells, unpublished).
- the hGHbp was conjugated as recommended by the supplier to a level of 1.7 ⁇ mol hGHbp/mg dry oxirane bead, as measured by binding of [ 125 I] hGH to the resin. Subsequently, any unreacted oxirane groups were blocked with BSA and Tris.
- BSA was similarly coupled to the beads.
- Buffer for adsorption and washing contained 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg/ml BSA, and 0.02% Tween 20.
- Elution buffers contained wash buffer plus 200 nM hGH or 0.2 M glycine (pH 2.1).
- Parental phage M13KO7 was mixed with hGH phagemid particles at a ratio of nearly 3000:1 (original mixture) and tumbled for 8-12 h with a 5 ⁇ l aliquot (0.2 mg of acrylamide beads) of either absorbent in a 50 ⁇ l volume at room temperature.
- the beads were pelleted by centrifugation and the supernate carefully removed.
- the beads were resuspended in 200 ⁇ l wash buffer and tumbled at room temperature for 4 hours (wash 1). After a second wash (wash 2), the beads were eluted twice with 200 nM hGH for 6-10 hours each (eluate 1, eluate 2).
- the final elution was with a glycine buffer (pH 2.1) for 4 hours to remove remaining hGH phagemid particles (eluate 3).
- Each fraction was diluted appropriately in 2YT media, mixed with fresh JM101, incubated at 37° C. for 5 minutes, and plated with 3 ml of 2YT soft agar on LB or LB carbenicillin plates.
- the gene III protein is composed of 410 residues divided into two domains that are separated by a flexible linker sequence (Armstrong, J., et al., FEBS Lett., 135:167-172, [1981]).
- the amino-terminal domain is required for attachment to the pili of E. coli , while the carboxyl-terminal domain is imbedded in the phage coat and required for proper phage assembly (Crissman, J. W., Smith, G. P., Virology, 132:445-455, [1984]).
- the signal sequence and amino-terminal domain of gene III was replaced with the stII signal and entire hGH gene (Chang et al.
- FIG. 1 by fusion to residue 198 in the carboxyl-terminal domain of gene III ( FIG. 1 ).
- the hGH-gene III fusion was placed under control of the lac promoter/operator in a plasmid (phGH-M13gIII; FIG. 1 ) containing the pBR322 ⁇ -lactamase gene and Col E1 replication origin, and the phage f1 intergenic region.
- the vector can be easily maintained as a small plasmid vector by selection on carbenicillin, which avoids relying on a functional gene III fusion for propagation.
- the plasmid can be efficiently packaged into virions (called phagemid particles) by infection with helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly.
- helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly.
- Phagemid infectivity titers based upon transduction to carbenicillin resistance in this system varied from 2-5 ⁇ 10 11 colony forming units (cfu)/ml.
- the titer of the M13KO7 helper phage in these phagemid stocks is ⁇ 10 10 plaque forming units (pfu)/ml.
- the titer of fusion phage displaying the hGH gene III fusion is about 2 ⁇ 5 ⁇ 10 10 /ml. This number is much greater than the titer of E. coli ( ⁇ 10 8 to 10 9 /ml) in the culture from which they are derived. Thus, on average every E. coli cell produces 10-100 copies of phage decorated with an hGH gene III fusion protein.
- FIG. 2 Immunoblot analysis ( FIG. 2 ) of the hGH-gene III phagemid show that hGH cross-reactive material comigrates with phagemid particles in agarose gels. This indicates that the hGH is tightly associated with phagemid particles.
- the hGH-gene III fusion protein from the phagemid particles runs as a single immuno-stained band showing that there is little degradation of the hGH when it is attached to gene III. Wild-type gene III protein is clearly present because about 25% of the phagemid particles are infectious. This is comparable to specific infectivity estimates made for wild-type M13 phage that are similarly purified (by CsCl density gradients) and concentrations estimated by UV absorbance (Smith, G. P. supra and Parmley, Smith supra) Thus, both wild-type gene III and the hGH-gene III fusion proteins are displayed in the phage pool.
- hGHbp The extracellular domain of the hGH receptor (hGHbp) (Fuh et al., supra) containing a free cysteino residue was efficiently coupled to these beads and phagemid particles showed very low non-specific binding to beads coupled only to bovine serum albumin (Table II).
- a fusion phagemid was constructed with an hGH mutant in which Arg64 was substituted with Ala (R64A).
- the R64A variant hormone is about 20-fold reduced in receptor binding affinity compared to hGH (Kd values of 7.1 nM and 0.34 nM, respectively [Cunningham, Wells, supra]).
- the titers of the R64A hGH-gene III fusion phagemid were comparable to those of wild-type hGH phagemid.
- the wild-type hGH phagemid was enriched from a mixture of the two phagemids plus M13KO7 by 8-fold relative to the phagemid R64A, and ⁇ 10 4 relative to M13KO7 helper phage.
- Binding selections were carried out using beads linked with BSA (control beads) or with the hGHbp (hGHbp beads) as described in Table II and the Materials and Methods After each step, plasmid DNA was isolated (Birnboim, H. C., Doly, J., Nucleic Acids Res., 7: 1513-1523, [1979]) from carbenicillin resistant colonies and analyzed by restriction analysis # to determine if it contained the wild-type hGH or the R64A hGH gene III fusion.
- hGH a 22 kD protein
- Protein-protein and antibody-antigen interactions are dominated by discontinuous epitopes (Janin, J., et al., J. Mol. Biol., 204:155-164, [1988]; Argos, P., Prot. Eng., 2:101-113, [1988]; Barlow, D. J., et al., Nature, 322:747-748, [1987]; add Davies, D. R., et al., J. Biol. Chem., 263:10541-10544, [1988]); that is the residues directly involved in binding are close in tertiary structure but separated by residues not involved in binding.
- the screening system presented here should allow one to analyze more conveniently protein-receptor interactions and isolate discontinuous epitopes in proteins with new and high affinity binding properties.
- a mutant of the hGH-gene III fusion protein was constructed using the method of Kunkel., et al. Meth. Enzymol. 154, 367-382 [1987].
- Template DNA was prepared by growing the plasmid pS0132 (containing the natural hGH gene fused to the carboxy-terminal half of M13 gene III, under control of the alkaline phosphatase promoter) in CJ236 cells with M13-K07 phage added as helper.
- Single-stranded, uracil-containing DNA was prepared for mutagenesis to introduce (1) a mutation in hGH which would greatly reduce binding to the hGH binding protein (hGHbp); and (2) a unique restriction site (KpnI) which could be used for assaying for—and selecting against—parental background phage.
- Oligonucleotide-directed mutagenesis was carried out using T7 DNA polymerase and the following oligodeoxy-nucleotide (SEQ ID NO:7): Gly Thr hGH codon: 178 179 5′-G ACA TTC CTG G G T A C C GTG CAG T-3′ ⁇ KpnI > This oligo introduces the KpnI site as shown, along with mutations (R178G, I179T) in hGH. These mutations are predicted to reduce binding of hGH to hGHbp by more than 30-fold. Clones from the mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. The resulting construct, to be used as a template for random mutagenesis, was designated pHO415. Random Mutagenesis within Helix-4 of hGH
- Codons 172, 174, 176, 178 were targeted for random mutagenesis in hGH, again using the method of Kunkel.
- Single-stranded template from pHO415 was prepared as above and mutagenesis was carried out using the following pool of oligos (SEQ ID NO:8): hGH codon: 172 174 5′- GC TTC AGG AAG GAC ATG GAC NNS GTC NNS ACA- Ile 176 178 179 - NNS CTG NNS A T C GTG CAG TGC CGC TCT GTG G-3′
- the mutagenesis products were extracted twice with phenol:chloroform (50:50) and ethanol precipitated with an excess of carrier tRNA to avoid adding salt that would confound the subsequent electroporation step.
- dsDNA double-stranded DNA
- pLIB1 double-stranded DNA
- the supernatant was spun again to remove any remaining cells, and the phage, designated phage pool ⁇ 1, were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl).
- Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p gene III fusion (hGH-g 3 ) plasmid, and plaque-forming units (PFU) for the M13-K07 helper phage.
- CFU colony-forming units
- PFU plaque-forming units
- BINDING An aliquot of phage pool ⁇ 1 (6 ⁇ 10 9 CFU, 6 ⁇ 10 7 PFU) was diluted 4.5-fold in buffer A (Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal) and mixed with a 5 ⁇ L suspension of oxirane-polyacrylamide beads coupled to the hGHbp containing a Ser237 Cys mutation (350 fmols) in a 1.5 mL silated polypropylene tube.
- buffer A Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal
- an equivalent aliquot of phage were mixed in a separate tube with beads that had been coated with BSA only.
- the phage were allowed to bind to the beads by incubating 3 hours at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were
- hGH ELUTION Phage/phagemid binding weakly to the beads were removed by stepwise elution with hGH. In the first step, the beads were rotated with buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted and resuspended in buffer A containing 20 nM hGH and rotated for 3 hours, then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH and rotated for 3 hours then pelleted.
- GLYCINE ELUTION To remove the tightest-binding phagemid (i.e. those still bound after the hGH washes), beads were suspended in Glycine buffer (1 M Glycine, pH 2.0 with HCl), rotated 2 hours and pelleted. The supernatant (fraction “G”; 200 ⁇ L) was neutralized by adding 30 ⁇ L of 1 M Tris base.
- the hGHbp-beads yielded 14 times as many CFU's. This reflects the enrichment of tight-binding hGH-displaying phagemid over nonspecifically-binding phagemid.
- Phage from library 1G ( FIG. 3 ) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained 30 times as many CFU's as fraction G eluted from BSA-beads in this selection. Again, an aliquot of fraction G was propagated in WJM101 cells to yield library 1G 2 (indicating that this library had been twice selected by glycine elution). Double-stranded DNA (pLIB 1G 2 ) was also prepared from this culture.
- phage Library 2 ( FIG. 3 ).
- Phagemid binding, elution, and propagation were carried out in successive rounds for phagemid derived from both pLIB 2 and pLIB 3 ( FIG. 3 ) as described above, except that (1) an excess (10-fold over CFU) of purified K07 phage (not displaying hGH) was added in the bead-binding cocktail, and (2) the hGH stepwise elutions were replaced with brief washings of buffer A alone. Also, in some cases, XL1-Blue cells were used for phagemid propagation.
- Residue 178-Arg is also the same as wild-type, but here, the codon selected from the library was MG instead of CGC as is found in wild-type hGH, even though the latter codon is also possible using the “NNS” codon set. Multiplicity of K07 Infection
- the multiplicity of infection of K07 infection is an important parameter in the propagation of recombinant phagemids.
- the K07 multiplicity of infection must be high enough to insure that virtually all cells transformed or transfected with phagemid are able to package new phagemid particles.
- the concentration of wild-type gene III in each cell should be kept high to reduce the possibility of multiple hGH-gene III fusion molecules being displayed on each phagemid particle, thereby reducing chelate effects in binding.
- the K07 multiplicity of infection is too high, the packaging of K07 will compete with that of recombinant phagemid.
- Phage pools are labelled as shown ( FIG. 3 ).
- the multiplicity of infection (moi) refers to the multiplicity of K07 infection (PFU/cells) in the propagation of phagemid.
- the enrichment of CFU over PFU is shown in those cases where purified K07 was added in the binding step.
- the ratio of CFU eluting from hGHbp-beads over CFU eluting from BSA-beads is shown.
- the fraction of KpnI-containing template (i.e., pH0415) remaining in the pool was determined by digesting dsDNA with KpnI plus EcoRI, running the products on a 1% agarose gel, and laser-scanning a negative of the ethidium bromide-stained DNA.
- Human growth hormone variants were produced by the method of the present invention using the phagemid described in FIG. 9 .
- Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis (Kunkel et al., Methods Enzymol. 154, 367-382 [1987]) of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter (Bass et al., Proteins 8, 309-314 [1990]) ( FIG. 9 ).
- Mutagenesis was carried out with the oligonucleotide 5′-GGC-AGC-TGT-GGC-T TC-TAG-A GT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduces a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH.
- pS0643 and derivatives can be grown in a amber-suppressor strain of E. coli , such as JM101 or XL1-Blue (Bullock et al., BioTechniques 5, 376-379 [1987]). Shown above is substitution of Glu at the amber codon which occurs in supE suppressor strains. Suppression with other amino acids is also possible in various available strains of E. coli well known and publically available.
- pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9.
- the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- pS0643 was mutated with the oligonucleotides (1) 5′-CGG-ACT-GGG-C AG-ATA-T TC-AAG-CAG-ACC-3′ (SEQ ID NO:13), which destroys the unique BglII site of pS0643; (2) 5′-CTC-AAG-AAC-TAC-G GG-TTA-CC C-TGA-CTG-CTT-CAG-GAA-GG-3′ (SEQ ID NO:14), which inserts a unique BstEII site, a single-base frameshift, and a non-amber stop codon (TGA); and (3) 5′-CGC-ATC-GTG-CAG-TGC- AGA-TCT -GTG-GAG-GGC-3′ (SEQ ID NO:15), which introduces a new BgIII site, to yield the starting vector, pH0509.
- Codons 172, 174, 176 and 178 of hGH were targeted for random mutagenesis because they all lie on or near the surface of hGH and contribute significantly to receptor-binding (Cunningham and Wells, Science 244, 1081-1085 [1989]); they all lie within a well-defined structure, occupying 2 “turns” on the same side of helix 4; and they are each substituted by at least one amino acid among known evolutionary variants of hGH.
- TAG amber
- the vector was prepared by digesting pH0509 with BstEII followed by BglII. The products were run on a 1% agarose gel and the large fragment excised, phenol-extracted, and ethanol precipitated. This fragment was treated with calf intestinal phosphatase (Boehringer), then phenol:chloroform extracted, ethanol precipitated, and resuspended for ligation with the mutagenic cassette.
- reaction products were again digested with BstEII, then phenol:chloroform extracted, ethanol precipitated and resuspended in water.
- a BstEII recognition site (GGTNACC) is created within cassettes which contain a G at position 3 of codon 172 and an ACC (Thr) codon at 174.
- dsDNA double-stranded DNA
- pH0529E the initial library
- phage pool ⁇ H0529E the initial library of phage
- Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p. Approximately 4.5 ⁇ 10 13 CFU were obtained from the starting library.
- hGHbp-beads Immobilized hGHbp (“hGHbp-beads”) was prepared as described (Bass et al., Proteins 8, 309-314 [1990]), except that wild-type hGHbp (Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]) was used.
- hPRLbp-beads Immobilized hPRLbp (“hPRLbp-beads”) was prepared as above, using the 211-residue extracellular domain of the prolactin receptor (Cunningham et al., Science 250, 1709-1712 [1990]).
- “Blank beads” were prepared by treating the oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) for 15 hours at 4° C.
- Buffer A PBS, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal
- Buffer B 50 mM tris pH 7.5, 10 mM MgCl 2 , 0.5% BSA, 0.05% Tween 20, 100 mM ZnCl 2
- Buffer C PBS, 0.5% BSA, 0.05% Tween 20, 0.01% thimerosal, 10 mM EDTA
- Binding selections were carried out according to each of the following paths: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+Zn 2+ ), (4) binding to hPRLbp-beads (+EDTA), (5) pre-adsorbing twice with hGHbp beads then binding the non-adsorbed fraction to hPRLbp-beads (“ ⁇ hGHbp, +hPRLbp” selection), or (6) pre-adsorbing twice with hPRLbp-beads then binding the non-adsorbed fraction to hGHbp-beads (“ ⁇ hPRLbp, +hGHbp” selection).
- Binding and elution of phage was carried out in each cycle as follows:
- BINDING An aliquot of hormone phage (typically 10 9 -10 10 CFU) was mixed with an equal amount of non-hormone phage (pCAT), diluted into the appropriate buffer (A, B, or C), and mixed with a 10 mL suspension of hGHbp, hPRLbp or blank beads in a total volume of 200 mL in a 1.5 mL polypropylene tube. The phage were allowed to bind to the beads by incubating 1 hour at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 ⁇ L and at room temperature.
- pCAT non-hormone phage
- WASHES The beads were spun 15 sec., and the supernatant was removed. To reduce the number of phage not specifically bound, the beads were washed 5 times by resuspending briefly in the appropriate buffer, then pelleting.
- hGH ELUTION Phage binding weakly to the beads were removed by elution with hGH. The beads were rotated with the appropriate buffer containing 400 nM hGH for 15-17 hours. The supernatant was saved as the “hGH elution” and the beads. The beads were washed by resuspending briefly in buffer and pelleting.
- GLYCINE ELUTION To remove the tightest-binding phage (i.e. those still bound after the hGH wash), beads were suspended in Glycine buffer (Buffer A plus 0.2 M Glycine, pH 2.0 with HCl), rotated 1 hour and pelleted. The supernatant (“Glycine elution”; 200 ⁇ L) was neutralized by adding 30 mL of 1 M Tris base and stored at 4° C.
- Phage binding, elution, and propagation were carried out in successive rounds, according to the cycle described above.
- the phage amplified from the hGH elution from hGHbp-beads were again selected on hGHbp-beads and eluted with hGH, then used to infect a new culture of XL1-Blue cells.
- Three to five rounds of selection and propagation were carried out for each of the selection procedures described above.
- Mutants of hGH were prepared from osmotically shocked cells by ammonium sulfate precipitation as described for hGH (Olson et al., Nature 293, 408-411 [1981]), and protein concentrations were measured by laser densitomoetry of Coomassie-stained SDS-polyacrylamide gel electrophoresis gels, using hGH as standard (Cunningham and Wells, Science 244, 1081-1085 [1989]).
- the binding affinity of each mutant was determined by displacement of 125 I hGH as described (Spencer et al., J. Biol. Chem. 263, 7862-7867 [1988]; Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]), using an anti-receptor monoclonal antibody (Mab263).
- Binding assays may be carried out for mutants selected for hPRLbp-binding. TABLE VII Competitive binding to hGHbp The selected pool in which each mutant was found is indicated as 1G (first glycine selection), 3G (third glycine selection), 3H (third hGH selection), 3* (third selection, not binding to hPRLbp, but binding to hGHbp). The number of times each mutant occurred among all sequenced clones is shown ( ).
- Kd(mut)/ Mutant Kd (nM) Kd(hGH) Pool KSYR (6) (SEQ ID NO: 84) 0.06 + 0.01 0.18 1G, 3G RSFR (SEQ ID NO: 85) 0.10 + 0.05 0.30 3G RAYR (SEQ ID NO: 86) 0.13 + 0.04 0.37 3* KTYK (2) (SEQ ID NO: 87) 0.16 + 0.04 0.47 H, 3G RSYR (3) (SEQ ID NO: 88) 0.20 + 0.07 0.58 1G, 3H, 3G KAYR (3) (SEQ ID NO: 89) 0.22 + 0.03 0.66 3G RFFR (2) (SEQ ID NO: 90) 0.26 + 0.05 0.76 3H KQYR (SEQ ID NO: 91) 0.33 + 0.03 1.0 3G KEFR wt (9) 0.34 + 0.05 1.0 3H, 3G, 3* RTYH (SEQ ID NO: 92) 0.68 + 0.17 2.0 3H QRYR (
- substitution of a particular amino acid has essentially the same effect independent of surrounding residues.
- substitution of F176Y in the background of 172R/174S reduces binding affinity by 2.0-fold (RSFR (SEQ ID NO:85) vs. RSYR (SEQ ID NO:88)).
- RSFR SEQ ID NO:85
- RSYR SEQ ID NO:88
- the binding affinity of the F176Y mutant is 2.9-fold weaker than the corresponding 176F mutant (KAFR; Cunningham and Wells, 1989).
- the binding constants determined for several selected mutants of hGH demonstrate non-additive effects of some amino acid substitutions at residues 172, 174, 176, and 178.
- the substitution E174S results in a mutant (KSYR (SEQ ID NO:84)) which binds hGHbp 3.7-fold tighter than the corresponding mutant containing E174A (KAYR (SEQ ID NO:89)).
- the effects of these E174 substitutions are reversed.
- the E174A mutant (RAYR (SEQ ID NO:86)
- RYR SEQ ID NO:88
- Example VIII Using the methods described in Example VIII, we targeted another region of hGH involved in binding to the hGHbp and/or hPRLbp, helix 1 residues 10, 14, 18, 21, for random mutagenesis in the phGHam-g3p vector (also known as pS0643; see Example VIII).
- phGHam-g3p the “amber” hGH-g3 construct
- Phage produced from both pS0132 S. Bass, R. Greene, J. A. Wells, Proteins 8, 309 (1990).
- phGHam-g3 were tested with three antibodies (Medix 2, 1B5.G2, and 5B7.C10) that are known to have biriding determinants near the carboxyl-terminus of hGH [B. C. Cunningham, P. Jhurani, P. Ng, J. A.
- Phagemid particles from phGHam-g3 reacted much more strongly with antibodies Medix 2, 1B5.G2, and 5B7.C10 than did phagemid particles from pS0132.
- binding of pS0132 particles was reduced by >2000-fold for both Medix 2 and 5B7.C10 and reduced by >25-fold for 1B5.G2 compared to binding to Medix 1.
- binding of phGHam-g3 phage was weaker by only about 1.5-fold, 1.2-fold, and 2.3-fold for the Medix 2, 1B5.G2, and 5B7.C10 antibodies, respectively, compared with binding to MEDIX 1.
- This library was constructed by cassette mutagenesis that fully mutated four residues at a time (see Example VIII) which utilized a mutated version of phGHam-g3 into which unique KpnI (at hGH codon 27) and XhoI (at hGH codon 6) restriction sites (underlined below) had been inserted by mutagenesis [T. A. Kunkel, J. D. Roberts, R. A. Zakour, Methods Enzymol.
- oligonucleotides 5′-GCC TTT GAC A GG TAC C AG GAG m G-3′ (SEQ ID NO:18) and 5′-CCA ACT ATA CCA CT C TCG AG G TCT ATT CGA TAA C-3′ (SEQ ID NO:19), respectively.
- the later oligo also introduced a+1 frameshift (italicized) to terminate translation from the starting vector and minimize wild-type background in the phagemid library. This strating vector was designated pH0508B.
- the helix 1 library which mutated hGH residues 10, 14, 18, 21, was constructed by ligating to the large XhoI-KpnI fragment of pH0508B a cassette made from the complementary oligonucleotides 5′-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3′ (SEQ ID NO:20) and 5′-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3′ (SEQ ID NO:21).
- the KpnI site was destroyed in the junction of the ligation product so that restriction enzyme digestion could be used for analysis of non-mutated background.
- the library contained at least 10 7 independent transformants so that if the library were absolutely random (10 6 different combinations of codons) we would have an average of about 10 copies of each possible mutated hGH gene. Restriction analysis using KpnI indicated that at least 80% of helix 1 library constructs contained the inserted cassette.
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII).
- hGHbp immobilized on oxirane-polyacrylamide beads
- Table VIII Four residues in helix 1 (F10, M14, H18, and H21) were similarly mutated and after 4 and 6 cycles a non-wild-type consensus developed (Table VIII).
- Position 10 on the hydrophobic face of helix 1 tended to be hydrophobic whereas positions 21 and 18 on the hydrophillic face tended were dominated by Asn; no obvious consensus was evident for position 14 (Table IX).
- the binding constants for these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, several mutants bind tighter to hGHbp than does wt-hGH.
- the helix 4b library was constructed in an attempt to further improve the helix 4 double mutant (E174S/F176Y) selected from the helix 4a library that we found bound tighter to the hGH receptor (see Example VIII). With the E174S/F176Y hGH mutant as the background starting hormone, residues were mutated that surrounded positions 174 and 176 on the hydrophilic face of helix 4 (R167, D171, T175 and I179).
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). After 6 cycles of binding a reasonably clear consensus developed (Table XI). Interestingly, all positions tended to contain polar residues, notably Ser, Thr and Asn (XII).
- the binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, the binding affinities of several helix-4-b mutants for hGHbp were tighter than that of wt-hGH Table XIII).
- the E174S/F176Y mutant binds 200-fold weaker to the hPRLbp than hGH.
- the E174T/F176Y/R178K and R167N/D171 S/E174S/F176Y/1179T mutants each bind >500-fold weaker to the hPRLbp than hGH.
- Hormone-Phagemid Selection Identifies the Information-Content of Particular Residues
- mutations learned through hormone-phagemid enrichment to improve binding can be combined by simple cutting and ligation of restriction fragments or mutagenesis to yield cumulatively optimized mutants of hGH.
- hormone phagemid enrichment can be carried out by one of several variations on the iterative enrichment approach: (1) random DNA libraries can be generated in each of two (or perhaps more) regions of the molecule by cassette or another mutagenesis method.
- a combined library can be created by ligation of restriction fragments from the two DNA libraries; (2) an hGH variant, optimized for binding by mutation in one region of the molecule, can be randomly mutated in a second region of the molecule as in the helix-4-b library example; (3) two or more random libraries can be partially selected for improved binding by hormone-phagemid enrichment; after this “roughing-in” of the optimized binding site, the still-partially-diverse libraries can be recombined by ligation of restriction fragments to generate a single library, partially diverse in two or more regions of the molecules, which in turn can be further selected for optimized binding using hormone-phagemid enrichment.
- Plasmid pDH 188 contains the DNA encoding the F ab portion of a humanized IgG antibody, called 4D5, that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR 101, and has been deposited with the ATCC in Rockville, Md.
- the plasmid was prepared as follows: the starting plasmid was pS0132, containing the alkaline phosphatase promoter as described above.
- the DNA encoding human growth hormone was excised and, after a series of manipulations to make the ends of the plasmid compatible for ligation, the DNA encoding 4D5 was inserted.
- the 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain, and contains at its 5′ end the DNA encoding the st II signal sequence. The second gene contains four portions: first, at its 5′ end is the DNA encoding the st II signal sequence.
- PEG polyethylene glycol
- electroporation Both polyethylene glycol (PEG) and electroporation were used to transform plasmids into SR101 cells.
- PEG competent cells were prepared and transformed according to the method of Chung and Miller ( Nucleic Acids Res. 16:3580 [1988]). Cells that were competent for electroporation were prepared, and subsequently transformed via electroporation according to the method of Zabarovsky and Winberg ( Nucleic Acids Res. 18:5912 [1990]). After placing the cells in 1 ml of the SOC media (described in Sambrook et al., supra), they were grown for 1 hour at 37° C. with shaking. At this time, the concentration of the cells was determined using light scattering at OD 600 .
- a titered K 7 phage stock was added to achieve an multiplicity of infection (MOI) of 100, and the phage were allowed to adhere to the cells for 20 minutes at room temperature. This mixture was then diluted into 25 mls of 2YT broth (described in Sambrook et al, supra) and incubated with shaking at 37° C. overnight. The next day, cells were pelleted by centrifugation at 5000 ⁇ g for 10 minutes, the supernatant was collected, and the phage particles were precipitated with 0.5 M NaCl and 4% PEG (final concentration) at room temperature for 10 minutes.
- MOI multiplicity of infection
- Phage particles were pelleted by centrifugation at 10,000 ⁇ g for 10 minutes, resuspended in 1 ml of TEN (10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl), and stored at 4° C.
- TEN 10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl
- phage particles were mixed with a 100-fold excess of K 7 helper phage and 1 ml of buffer A. This mixture was divided into two 0.5 ml aliquots; one of which was applied to ECD coated wells, and the other was applied to BSA coated wells. The plates were incubated at room temperature while shaking for one to three hours, and were then washed three times over a period of 30 minutes with 1 ml aliquots of buffer A.
- Elution of the phage from the plates was done at room temperature by one of two methods: 1) an initial overnight incubation of 0.025 mg/ml purified Mu4D5 antibody (murine) followed by a 30 minute incubation with 0.4 ml of the acid elution buffer (0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20), or 2) an incubation with the acid elution buffer alone. Eluates were then neutralized with 1 M Tris base, and a 0.5 ml aliquot of TEN was added. These samples were then propagated, titered, and stored at 4° C.
- the acid elution buffer 0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20
- the affinity of h4D5 F ab fragments and F ab phage for the ECD antigen was determined using a competitive receptor binding RIA (Burt, D. R., Receptor Binding in Drug Research . O'Brien, R. A. (Ed.). pp. 3-29, Dekker, New York [1986]).
- the ECD antigen was labeled with 125 -Iodine using the sequential chloramine-T method (De Larco, J. E. et al., J. Cell. Physiol. 109:143-152 [1981]) which produced a radioactive tracer with a specific activity of 14 ⁇ Ci/ ⁇ g and incorporation of 0.47 moles of Iodine per mole of receptor.
- a series of 0.2 ml solutions containing 0.5 ng (by ELISA) of F ab or F ab phage, 50 nCi of 125 I ECD tracer, and a range of unlabeled ECD amounts (6.4 ng to 3277 ng) were prepared and incubated at room temperature overnight.
- the labeled ECD-F ab or ECD-F ab phage complex was separated from the unbound labeled antigen by forming an aggregate complex induced by the addition of an anti-human IgG (Fitzgerald 40-GH23) and 6% PEG 8000.
- the complex was pelleted by centrifugation (15,000 ⁇ g for 20 minutes) and the amount of labeled ECD (in cpm) was determined by a gamma counter.
- the dissociation constant (K d ) was calculated by employing a modified version of the program LIGAND (Munson, P. and Rothbard, D., Anal. Biochem. 107:220-239 [1980]) which utilizes Scatchard analysis (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672 [1949]).
- the Kd values are shown in FIG. 13 .
- Murine 4D5 antibody was labeled with 125-I to a specific activity of 40-50 ⁇ Ci/ ⁇ g using the lodogen procedure. Solutions containing a constant amount of labeled antibody and increasing amounts of unlabeled variant F ab were prepared and added to near confluent cultures of SK-BR-3 cells grown in 96-well microtiter dishes (final concentration of labeled antibody was 0.1 nM). After an overnight incubation at 4° C., the supernatant was removed, the cells were washed and the cell associated radioactivity was determined in a gamma counter. K d values were determined by analyzing the data using a modified version of the program LIGAND (Munson, P. and Rothbard, D., supra)
- hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix variants was isolated and digested with the restriction enzymes EcoRI and BstXI. The large fragment from each helix-4-b variant was then isolated and ligated with the small fragment from each helix-1 variant to yield the new two-helix variants shown in Table XIII. All of these variants also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- hGH-phagemid double-stranded DNA from each of the one-helix library pools (selected for 0, 2, or 4 rounds) was isolated and digested with the restriction enzymes AccI and BstXI.
- the large fragment from each helix-1 variant pool was then isolated and ligated with the small fragment from each helix-4-b variant pool to yield the three combinatorial libraries pH0707A (unselected helix 1 and helix 4b pools, as described in examples IX and X), pH0707B (twice-selected helix-1 pool with twice-selected helix-4-b pool), and pH0707C (4-times selected helix-1 pool with 4-times selected helix-4-b pool).
- Duplicate ligations were also set up with less DNA and designated as pH0707D, pH0707E, and pH0707F, corresponding to the 0-, 2-, and 4-round starting libraries respectively. All of these variant pools also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- the ligation products pH0707A-F were processed and electro-transformed into XL1-Blue cells as described (Example VIII). Based on colony-forming units (CFU), the number of transformants obtained from each pool was as follows: 2.4 ⁇ 10 6 from pH0707A, 1.8 ⁇ 10 6 from pH0707B, 1.6 ⁇ 10 6 from pH0707C, 8 ⁇ 10 5 from pH0707D, 3 ⁇ 10 5 from pH0707E, and 4 ⁇ 10 5 from pH0707F.
- hGH-phagemid particles were prepared and selected for hGHbp-binding over 2 to 7 cycles as described in Example VIII.
- the pharmacological properties of a protein may be dependent on binding affinity or on k on or k off , depending on the detailed mechanism of action.
- phagemid particles from the pH0707B pool were incubated with immobilized hGHbp for only 1 minute, then washed six times with 1 mL of binding buffer; the hGH-wash step was omitted; and the remaining hGH-phagemid particles were eluted with a pH2 (0.2M glycine in binding buffer) wash. Enrichment of hGH-phagemid particles over non-displaying particles indicated that even with a short binding period and no cognate-ligand (hGH) challenge, hGH-phagemid binding selection sorts tight-binding variants out of a randomized pool.
- the binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9 or 34B8, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII) in a radio-immunoprecipitation assay.
- Table XIII below, all the variants have glutamate 174 replaced by serine 174 and phenylalanine 176 replaced by tyrosine 176 (E174S and F1176Y) plus the additional substitutions as indicated at hGH amino acid positions 10, 14, 18, 21, 167, 171, 175 and 179.
- hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XIV contain two background mutations (E174S/F176Y). hGH-phagemid pools from the libraries pH0707A (Part A), pH0707B and pH0707E (Part B), or pH0707C (Part C) were sorted for 2 to 7 cycles for binding to hGHbp. The number P indicates the fractional occurrence of each variant type among the set of clones sequenced from each pool. TABLE XIV hGH variants from hormone-phagemid binding selection of combinatorial libraries.
- Helix 1 Helix 4 wild-type residue P Variant F10 M14 H18 H21 R167 D171 T175 I179 Part A: 4 cycles: 0.60 H0714A.1 H G N N N S T N 0.40 H0714A.4 A N D A N N T N* Part B: 2 cycles: 0.13 H0712B.1 F S F G H S T T 0.13 H0712B.2 H Q T S A D N S 0.13 H0712B.4 H G N N N A T T 0.13 H0712B.5 F S F L S D T T 0.13 H0712B.6 A S T N R D T I 0.13 H0712B.7 Q Y N N H S T T 0.13 H0712B.8 W G S S R D T I 0.13 H0712E.1 F L S S K N T V 0.13 H0712E.2 W N N S H S T T 0.13 H0712E.3 A N A S N S T T 0.13 H0712E.4 P S D N R D T I 0.
- hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XV contain two background mutations (E174S/F176Y). The number P is the fractional occurrence of a given variant among all clones sequenced after 4 cycles of rapid-binding selection.
- binding constants were measured by competitive displacement of 125 I-labelled hormone H0650BD or labelled hGH using hGHbp (1-238) and either Mab5 or Mab263.
- the variant H0650BD appears bind more than 30-fold tighter than wild-type hGH.
- TABLE XVI Equilibrium binding constants of selected hGH variants.
- the plasmid pS0132 contains the gene for hGH fused to the residue Pro198 of the gene III protein with the insertion of an extra glycine residue.
- This plasmid may be used to produce hGH-phage particles in which the hGH-gene III fusion product is displayed monovalently on the phage surface (Example IV).
- the fusion protein comprises the entire hGH protein fused to the carboxy terminal domain of gene III via a flexible linker sequence.
- subtilisin BPN′ a genetically engineered variant of subtilisin BPN′.
- A64SAL subtilisin contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly restricted so that certain histidine-containing substrates are preferentially hyrdrolysed (Carter et al., Science 237:394-399 (1987)).
- the sequence of the linker region in pS0132 was mutated to create a substrate sequence for A64SAL subtilisin, using the oligonucleotide 5′-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3′ (SEQ ID NO:27).
- Phagemid particles derived from pS0132 and pS0640 were constructed as described in Example I.
- a 10 ⁇ l aliquot of each phage pool was separately mixed with 300 of oxirane beads (prepared as described in Example II) in 100 ⁇ l of buffer comprising 20 mM Tris-HCl pH 8.6 and 2.5M NaCl.
- the binding and washing steps were performed as described in example VII.
- the beads were then resuspended in 400 ⁇ l of the same buffer, with or without 50 nM of A64SAL subtilisin. Following incubation for 10 minutes, the supernatants were collected and the phage titres (cfu) measured.
- Table XVII shows that approximately 10 times more substrate-containing phagemid particles (pS0640) were eluted in the presence of enzyme than in the absence of enzyme, or than in the case of the non-substrate phagemids (pS0132) in the presence or absence of enzyme. Increasing the enzyme, phagemid or bead concentrations did not improve this ratio.
- hGH human growth hormone
- a tight-binding variant of hGH was introduced in place of the wild-type hGH gene in pS0132 and pS0640.
- the hGH variant used was as described in example XI (pH0650bd) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr.
- Binding COSTAR 12-well tissue culture plates were coated for 16 hours with 0.5 ml/well 2 ug/ml hGHbp in sodium carbonate buffer pH 10.0. The plates were then incubated with 1 ml/well of blocking buffer (phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen) for 2 hours and washed in an assay buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 100 mM NaCl. Phagemids were again prepared as described in Example I: the phage pool was diluted 1:4 in the above assay buffer and 0.5 ml of phage incubated per well for 2 hours.
- blocking buffer phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen
- Table XVII shows that there was a dramatic increase in the ratio of specifically eluted substrate-phagemid particles compared to the method previously described for pS0640 and pS0132. It is likely that this is due to the fact that the tight-binding hGH mutant has a significantly slower off-rate for binding to hGH binding protein compared to wild-type hGH.
- Example XIII We sought to employ the selective enrichment procedure described in Example XIII to identify good substrate sequences from a library of random substrate sequences.
- This new construct was designated pDM0253 (The actual sequence of pDM0253 is 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC- C CC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:29), where the underlined base substitution is due to a spurious error in the mutagenic oligonucleotide).
- the tight-binding hGH variant described in example was introduced by exchanging a fragment from pDM0411 (example XIII) The resulting library vector was designated pDM0454.
- pDM0454 was digested with ApaI followed by SalI, then precipitated with 13% PEG 8000+10 mM MgCl 2 , washed twice in 70% ethanol and resuspended This efficiently precipitates the vector but leaves the small Apa-Sal fragment in solution (Pai thankar, K. R. and Prasad, K. S, N., Nucleic Acids Research 19:1346).
- the product was run on a 1% agarose gel and the ApaI-SalI digested vector excised, purified using a Bandprep kit (Pharmacia) and resuspended for ligation with the mutagenic cassette.
- the cassette to be inserted contained a DNA sequence similar to that in the linker region of pS0640 and pDM0411, but with the codons for the histidine and tyrosine residues in the substrate sequence replaced by randomised codons.
- the oligonucleotides used in the mutagenic cassettes were: 5′-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CM-3′ (coding strand) (SEQ ID NO:30) and 5′-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3′ (non-coding strand) (SEQ ID NO:31).
- This cassette also destroys the SalI site, so that digestion with SalI may be used to reduce the vector background.
- oligonucleotides were not phosphorylated before insertion into the Apa-Sal cassette site, as it was feared that subsequent oligomerisation of a small population of the cassettes may lead to spurious results with multiple cassette inserts.
- the reaction products were phenol:chloroform extracted, ethanol precipitated and resuspended in water. Initially, no digestion with SalI to reduce the background vector was performed. Approximately 200 ng was electroporated into XL-1 blue cells and a phagemid library was prepared as described in example VIII.
- the selection procedure used was identical to that described for pDM0411 and pDM0390 in example XIII. After each round of selection, the eluted phage were propagated by transducing a fresh culture of XL-1 blue cells and propagating a new phagemid library as described for hGH-phage in example VIII. The progress of the selection procedure was monitored by measuring eluted phage titres and by sequencing individual clones after each round of selection.
- Table A shows the successive phage titres for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection.
- Tables B1 and B2 shows the sequences of isolates obtained after round 2 and round 3 of selection. After 2 rounds of selection, there is clearly a high incidence of histidine residues. This is exactly what is expected: as described in example XIII, A64SAL subtilisin requires a histidine residue in the substrate as it employs a substrate-assisted catalytic mechanism. After 3 rounds of selection, each of the 10 clones sequenced has a histidine in the randomised cassette. Note, however, that 2 of the sequences are of pDM0411, which was not present in the starting library and is therefore a contaminant.
- Colony forming units were estimated by plating out 10 ⁇ l of 10-fold dilutions of phage on 10 ⁇ l spots of XL-1 blue cells, on LB agar plates containing 50 ⁇ g/ml carbenicillin ROUND 1
- Starting library 3 ⁇ 10 12 cfu/ml LIBRARY: +500 nM A64SAL 4 ⁇ 10 3 cfu/10 ⁇ l no enzyme 3 ⁇ 10 3 cfu/10 ⁇ l pDM0411: +500 nM A64SAL 2 ⁇ 10 6 cfu/10 ⁇ l (control) no enzyme 8 ⁇ 10 3 cfu/10 ⁇ l ROUND 2 Round 1 library: 7 ⁇ 10 12 cfu/ml LIBRARY: +500 nM A64SAL 3 ⁇ 10 4 cfu/10 ⁇ l no enzyme 6 ⁇ 10 3 cfu/10 ⁇ l
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Endocrinology (AREA)
- Wood Science & Technology (AREA)
- Hematology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Computational Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules is provided. The method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disclosed are preferred phagemid expression vectors and selected human growth hormone variants.
Description
- This application is a continuation application of Ser. No. 11/199,062 filed Aug. 8, 2005, which is a continuation of application Ser. No. 09/717,641 filed Nov. 21, 2000; which is a continuation of application Ser. No. 08/922,345 filed Sep. 3, 1997; which is a continuation of application Ser. No. 08/463,587 filed Jun. 5, 1995, now issued as U.S. Pat. No. 5,821,047; which is a divisional of application Ser. No. 08/050,058 filed Apr. 30, 1993, now issued as U.S. Pat. No. 5,750,373; which is a 371 of International Application No. PCT/US91/09133 filed Dec. 3, 1991; which is a continuation-in-part of application Ser. No. 07/743,614 filed Aug. 9, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/715,300 filed Jun. 14, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/683,400 filed Apr. 10, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07/621,667 filed Dec. 3, 1990, now abandoned. The contents of these applications are incorporated herein by reference.
- This invention relates to the preparation and systematic selection of novel binding proteins having altered binding properties for a target molecule. Specifically, this invention relates to methods for producing foreign polypeptides mimicking the binding activity of naturally occurring binding partners. In preferred embodiments, the invention is directed to the preparation of therapeutic or diagnostic compounds that mimic proteins or nonpeptidyl molecules such a hormones, drugs and other small molecules, particularly biologically active molecules such as growth hormone.
- Binding partners are substances that specifically bind to one another, usually through noncovalent interactions. Examples of binding partners include ligand-receptor, antibody-antigen, drug-target, and enzyme-substrate interactions. Binding partners are extremely useful in both therapeutic and diagnostic fields.
- Binding partners have been produced in the past by a variety of methods including; harvesting them from nature (e.g., antibody-antigen, and ligand-receptor pairings) and by adventitious identification (e.g. traditional drug development employing random screening of candidate molecules). In some instances these two approaches have been combined. For example, variants of proteins or polypeptides, such as polypeptide fragments, have been made that contain key functional residues that participate in binding. These polypeptide fragments, in turn, have been derivatized by methods akin to traditional drug development. An example of such derivitization would include strategies such as cyclization to conformationally constrain a polypeptide fragment to produce a novel candidate binding partner.
- The problem with prior art methods is that naturally occurring ligands may not have proper characteristics for all therapeutic applications. Additionally, polypeptide ligands may not even be available for some target substances. Furthermore, methods for making non-naturally occurring synthetic binding partners are often expensive and difficult, usually requiring complex synthetic methods to produce each candidate. The inability to characterize the structure of the resulting candidate so that rational drug design methods can be applied for further optimization of candidate molecules further hampers these methods.
- In an attempt to overcome these problems, Geysen (Geysen, Immun. Today. 6:364-369 [1985]); and (Geysen et al., Mol. Immun., 23:709-715 [1986]) has proposed the use of polypeptide synthesis to provide a framework for systematic iterative binding partner identification and preparation. According to Geysen et al., Ibid, short polypeptides, such as dipeptides, are first screened for the ability to bind to a target molecule. The most active dipeptides are then selected for an additional round of testing comprising linking, to the starting dipeptide, an additional residue (or by internally modifying the components of the original starting dipeptide) and then screening this set of candidates for the desired activity. This process is reiterated until the binding partner having the desired properties is identified.
- The Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure. As rounds of iterative selection progress, random interactions accelerate among the various substituent groups of the polypeptide so that a true random population of interactive molecules having reproducible higher order structure becomes less and less attainable. For example, interactions between side chains of amino acids, which are sequentially widely separated but which are spatially neighbors, freely occur. Furthermore, sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule. Such complex interactions are facilitated by the flexibility of the polyamide backbone of the polypeptide candidates. Additionally, candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.
- A final problem with the iterative polypeptide method of Geysen is that, at present, there are no practical methods with which a great diversity of different peptides can be produced, screened and analyzed. By using the twenty naturally occurring amino acids, the total number of all combinations of hexapeptides that must be synthesized is 64,000,000. Even having prepared such a diversity of peptides, there are no methods available with which mixtures of such a diversity of peptides can be rapidly screened to select those peptides having a high affinity for the target molecule. At present, each “adherent” peptide must be recovered in amounts large enough to carry out protein sequencing.
- To overcome many of the problems inherent in the Geysen approach, biological selection and screening was chosen as an alternative. Biological selections and screens are powerful tools to probe protein function and to isolate variant proteins with desirable properties (Shortle, Protein Engineering, Oxender and Fox, eds., A.R. Liss, Inc., NY, pp. 103-108 [1988]) and Bowie et al., Science, 247:1306-1310 [1990)]. However, a given selection or screen is applicable to only one or a small number of related proteins.
- Recently, Smith and coworkers (Smith, Science, 228: 1315-1317 [1985]) and Parmley and Smith, Gene, 73:305-318 [1985] have demonstrated that small protein fragments (10-50 amino acids) can be “displayed” efficiently on the surface of filamentous phage by inserting short gene fragments into gene III of the fd phage (“fusion phage”). The gene III minor coat protein (present in about 5 copies at one end of the virion) is important for proper phage assembly and for infection by attachment to the pili of E. coli (see Rasched et al., Microbiol. Rev., 50: 401-427 [1986]). Recently, “fusion phage” have been shown to be useful for displaying short mutated peptide sequences for identifying peptides that may react with antibodies (Scott et al., Science 249: 386-390, [1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A 87: 6378-6382, [1990]) or a foreign protein (Devlin et al, Science, 249: 404-406 [1990]).
- There are, however, several important limitations in using such “fusion phage” to identify altered peptides or proteins with new or enhanced binding properties. First, it has been shown (Parmley et al, Gene, 73: 305-318, [1988]) that fusion phage are useful only for displaying proteins of less than 100 and preferably less than 50 amino acid residues, because large inserts presumably disrupt the function of gene III and therefore phage assembly and infectivity. Second, prior art methods have been unable to select peptides from a library having the highest binding affinity for a target molecule. For example, after exhaustive panning of a random peptide library with an anti-β endorphin monoclonal antibody, Cwirla and co-workers could not separate moderate affinity peptides (Kd˜10 μM) from higher affinity peptides (Kd˜0.4 μM) fused to phage. Moreover, the parent β-endorphin peptide sequence which has very high affinity (Kd˜7 nM), was not panned from the epitope library.
- Ladner WO 90/02802 discloses a method for selecting novel binding proteins displayed on the outer surface of cells and viral particles where it is contemplated that the heterologous proteins may have up to 164 amino acid residues. The method contemplates isolating and amplifying the displayed proteins to engineer a new family of binding proteins having desired affinity for a target molecule. More specifically, Ladner discloses a “fusion phage” displaying proteins having “initial protein binding domains” ranging from 46 residues (crambin) to 164 residues (T4 lysozyme) fused to the M13 gene III coat protein. Ladner teaches the use of proteins “no larger than necessary” because it is easier to arrange restriction sites in smaller amino acid sequences and prefers the 58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI). Small fusion proteins, such as BPTI, are preferred when the target is a protein or macromolecule, while larger fusion proteins, such as T4 lysozyme, are preferred for small target molecules such as steroids because such large proteins have clefts and grooves into which small molecules can fit. The preferred protein, BPTI, is proposed to be fused to gene III at the site disclosed by Smith et al. or de la Cruz et al, J. Biol. Chem., 263: 4318-4322 [1988], or to one of the terminii, along with a second synthetic copy of gene III so that “some” unaltered gene III protein will be present. Ladner does not address the problem of successfully panning high affinity peptides from the random peptide library which plagues the biological selection and screening methods of the prior art.
- Human growth hormone (hGH) participates in much of the regulation of normal human growth and development. This 22,000 dalton pituitary hormone exhibits a multitude of biological effects including linear growth (somatogenesis), lactation, activation of macrophages, insulin-like and diabetogenic effects among others (Chawla, R, K. (1983) Ann. Rev. Med. 34, 519; Edwards, C. K. et al. (1988) Science 239, 769; Thorner, M. O., et al. (1988) J. Clin. Invest. 81, 745). Growth hormone deficiency in children leads to dwarfism which has been successfully treated for more than a decade by exogenous administration of hGH. hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants or growth hormone (Nicoll, C. S., et al, (1986)
Endocrine Reviews 7, 169). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W., et al, [1987]Nature 330, 537) or prolactin receptor (Boutin, J. M., et al, [1988] Ce: 53, 69). The cloned gene for hGH has been expressed in a secreted form in Escherichia coli (Chang, C. N., et al, [l987]Gene 55, 189) and its DNA and amino acid sequence has been reported (Goeddel, et al, [1979] Nature 281, 544; Gray, et al., [1985] Gene 39, 247). The three-dimensional structure of hGH is not available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S., et al., [1987] Proc. Natl. Acad. Sci. USA 84, 6434). Human growth hormone's receptor and antibody epitopes have been identified by homolog-scanning mutagenesis (Cunningham et al., Science 243: 1330, 1989). The structure of novel amino terminal methionyl bovine growth hormone containing a spliced-in sequence of human growthhormone including histidine 18 and histidine 21 has been shown (U.S. Pat. No. 4,880,910) - Human growth hormone (hGH) causes a variety of physiological and metabolic effects in various animal models including linear bone growth, lactation, activation of macrophages, insulin-like and diabetogenic effects and others (R. K. Chawla et al., Annu. Rev. Med. 34, 519 (1983); O. G. P. Isaksson et al., Annu. Rev. Physiol. 47, 483 (1985); C. K. Edwards et al., Science 239, 769 (1988); M. O. Thorner and M. L. Vance, J. Clin. Invest. 82, 745 (1988); J. P. Hughes and H. G. Friesen, Ann. Rev. Physiol. 47, 469 (1985)). These biological effects derive from the interaction between hGH and specific cellular receptors.
- Accordingly, it is an object of this invention to provide a rapid and effective method for the systematic preparation of candidate binding substances.
- It is another object of this invention to prepare candidate binding substances displayed on surface of a phagemid particle that are conformationally stable.
- It is another object of this invention to prepare candidate binding substances comprising fusion proteins of a phage coat protein and a heterologous polypeptide where the polypeptide is greater than 100 amino acids in length and may be more than one subunit and is displayed on a phagemid particle where the polypeptide is encoded by the phagemid genome.
- It is a further object of this invention to provide a method for the preparation and selection of binding substances that is sufficiently versatile to present, or display, all peptidyl moieties that could potentially participate in a noncovalent binding interaction, and to present these moieties in a fashion that is sterically confined.
- Still another object of the invention is the production of growth hormone variants that exhibit stronger affinity for growth hormone receptor and binding protein.
- It is yet another object of this invention to produce expression vector phagemids that contain a suppressible termination codon functionally located between the heterologous polypeptide and the phage coat protein such that detectable fusion protein is produced in a host suppressor cell and only the heterologous polypeptide is produced in a non-suppressor host cell.
- Finally, it is an object of this invention to produce a phagemid particle that rarely displays more than one copy of candidate binding proteins on the outer surface of the phagemid particle so that efficient selection of high affinity binding proteins can be achieved.
- These and other objects of this invention will be apparent from consideration of the invention as a whole.
- These objectives have been achieved by providing a method for selecting novel binding polypeptides comprising: (a) constructing a replicable expression vector comprising a first gene encoding a polypeptide, a second gene encoding at least a portion of a natural or wild-type phage coat protein wherein the first and second genes are heterologous, and a transcription regulatory element operably linked to the first and second genes, thereby forming a gene fusion encoding a fusion protein; (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids; (c) transforming suitable host cells with the plasmids; (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and (g) separating the phagemid particles that bind from those that do not. Preferably, the method further comprises transforming suitable host cells with recombinant phagemid particles that bind to the target molecule and repeating steps (d) through (g) one or more times.
- Additionally, the method for selecting novel binding proteins where the proteins are composed of more than one subunit is achieved by selecting novel binding peptides comprising constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein; mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors; transforming suitable host cells with the vectors; infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle; contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and separating the phagemid particles that bind from those that do not.
- Preferably in the method of this invention the plasmid is under tight control of the transcription regulatory element, and the culturing conditions are adjusted so that the amount or number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particle is less than about 1%. Also preferably, amount of phagemid particles displaying more than one copy of the fusion protein is less than 10% the amount of phagemid particles displaying a single copy of the fusion protein. Most preferably the amount is less than 20%.
- Typically, in the method of this invention, the expression vector will further contain a secretory signal sequences fused to the DNA encoding each subunit of the polypeptide, and the transcription regulatory element will be a promoter system. Preferred promoter systems are selected from; Lac Z, λPL, TAC, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
- Also typically, the first gene will encode a mammalian protein, preferably the protein will be selected from; human growth hormone (hGH), N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, such as betalactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, receptors for hormones or growth factors; integrin, thrombopoietin, protein A or D, rheumatoid factors, nerve growth factors such as NGF-β, platelet-growth factor, transforming growth factors (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferons such as interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF, interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins such as GP120, GP140, atrial natriuretic peptides A, B or C, immunoglobulins, and fragments of any of the above-listed proteins.
- Preferably the first gene will encode a polypeptide of one or more subunits containing more than about 100 amino acid residues and will be folded to form a plurality of rigid secondary structures displaying a plurality of amino acids capable of interacting with the target. Preferably the first gene will be mutated at codons corresponding to only the amino acids capable of interacting with the target so that the integrity of the rigid secondary structures will be preserved.
- Normally, the method of this invention will employ a helper phage selected from; M13KO7, M13R408, M13-VCS, and
Phi X 174. The preferred helper phage is M13KO7, and the preferred coat protein is the M13 Phage gene III coat protein. The preferred host is E. coli, and protease deficient strains of E. coli. Novel hGH variants selected by the method of the present invention have been detected. Phagemid expression vectors were constructed that contain a suppressible termination codon functionally located between the nucleic acids encoding the polypeptide and the phage coat protein. -
FIG. 1 . Strategy for displaying large proteins on the surface of filamentous phage and enriching for altered receptor binding properties. A plasmid, phGH-M13gIII was constructed that fuses the entire coding sequence of hGH to the carboxyl terminal domain of M13 gene III. Transcription of the fusion protein is under control of the lac promoter/operator sequence, and secretion is directed by the stII signal sequence. Phagemid particles are produced by infection with the “helper” phage, M13KO7, and particles displaying hGH can be enriched by binding to an affinity matrix containing the hGH receptor. The wild-type gene III (derived from the M13KO7 phage) is diagramed by 4-5 copies of the multiple arrows on the tip of the phage, and the fusion protein (derived from the phagemid, phGH-M13gIII) is indicated schematically by the folding diagram of hGH replacing the arrow head. -
FIG. 2 . Immunoblot of whole phage particles shows that hGH comigrates with phage. Phagemid particles purified in a cesium chloride gradient were loaded into duplicate wells and electrophoresed through a 1% agarose gel in 375 mM Tris, 40 mM glycine pH 9.6 buffer. The gel was soaked in transfer buffer (25 mM Tris, pH 8.3, 200 mM glycine, 20% methanol) containing 2% SDS and 2% β-mercaptoethanol for 2 hours, then rinsed in transfer buffer for 6 hours. The proteins in the gel were then electroblotted onto immobilon membranes (Millipore). The membrane containing one set of samples was stained with Coomassie blue to show the position of the phage proteins (A). The duplicate membrane was immuno-stained for hGH by reacting the membrane with polyclonal rabbit anti-hGH antibodies followed by reaction with horseradish peroxidase conjugated goat anti-rabbit IgG antibodies (B).Lane 1 contains the M13KO7 parent phage and is visible only in the Coomassie blue stained membrane, since it lacks hGH.Lanes -
FIG. 3 . Summary diagram of steps in the selection process for an hGH-phage library randomized atcodons Library 1. An aliquot (approximately 2%) fromLibrary 1 was used directly in an initial selection round as described in the text to yieldLibrary 1G. Meanwhile, double-stranded DNA (dsDNA) was prepared from Library I, digested with restriction enzyme KpnI to eliminate template background, and electrotransformed into WJM101 to yieldLibrary 2. Subsequent rounds of selection (or KpnI digestion, shaded boxes) followed by phagemid propagation were carried out as indicated by the arrows, according to the procedure described in the text. Four independent clones fromLibrary 4G4 and four independent clones fromLibrary 5G6 were sequenced by dideoxy sequencing. All of these clones had the identical DNA sequence, corresponding to the hGH mutant (Glu 174 Ser,Phe 176 Tyr). -
FIG. 4 . Structural model of hGH derived from a 2.8 Å folding diagram of porcine growth hormone determined crystallographically. Location of residues in hGH that strongly modulate its binding to the hGH-binding protein are within the shaded circle. Alanine substitutions that cause a greater than tenfold reduction (●), a four- to tenfold reduction (●), or increase (◯), or a two- to fourfold reduction (●), in binding affinity are indicated. Helical wheel projections in the regions of α-helix reveal their amphipathic quality. Blackened, shaded, or nonshaded residues are charged, polar, or nonpolar, respectively. In helix-4 the most important residues for mutation are on the hydrophilic face. -
FIG. 5 . Amino acid substitutions atpositions rounds positions position 176; and S, T, A, and other residues occur atposition 174. -
FIG. 6 . Sequences from phage selected on hPRLbp-beads in the presence of zinc. The notation is as described inFIG. 5 . Here, the convergence of sequences is not predictable, but there appears to be a bias towards hydrophobic sequences under the most stringent (Glycine) selection conditions; L W and P residues are frequently found in this pool. -
FIG. 7 . Sequences from phage selected on hPRLbp-beads in the absence of zinc. The notation is as described inFIG. 5 . In contrast to the sequences ofFIG. 6 , these sequences appear more hydrophilic. After 4 rounds of selection using hGH elution, two clones (ANHQ (SEQ ID NO:45), and TLDT/171V (SEQ ID NO:108)) dominate the pool. -
FIG. 8 . Sequences from phage selected on blank beads. The notation is as described inFIG. 5 . After three rounds of selection with glycine elution, no siblings were observed and a background level of non-functional sequences remained. -
FIG. 9 . Construction of phagemid fl ori from pHO415. This vector for cassette mutagenesis and expression of the hGH-gene III fusion protein was constructed as follows. Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter. Mutagenesis was carried out with theoligonucleotide 5′-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduced a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH. -
FIG. 10 . A. Diagram of plasmid pDH188 insert containing the DNA encoding the light chain and heavy chain (variable and constant domain 1) of the Fab humanized antibody directed to the HER-2 receptor. VL and VH are the variable regions for the light and heavy chains, respectively. Ck is the constant region of the human kappa light chain. CH1G1 is the first constant region of thehuman gamma 1 chain. Both coding regions start with the bacterial st II signal sequence. B. A schematic diagram of the entire plasma pDH188 containing the insert described in 5A. After transformation of the plasmid into E. coli SR101 cells and the addition of helper phage, the plasmid is packaged into phage particles. Some of these particles display the Fab-p III fusion (where p III is the protein encoded by the M13 gene III DNA). The segments in the plasmid figure correspond to the insert shown in 5A. -
FIG. 11A through C are collectively referred to here asFIG. 11 . The nucleotide (Seq. ID No:24) sequence of the DNA encoding the 4D5 Fab molecule expressed on the phagemid surface. The amino acid sequence of the light chain is also shown (Seq. ID No:25), as is the amino acid sequence of the heavy chain p III fusion (Seq. ID No:26). -
FIG. 12 . Enrichment of wild-type 4D5 Fab phagemid from variant Fab phagemid. Mixtures of wild-type phagemid and variant 4D5 Fab phagemid in a ratio of 1:1,000 were selected on plates coated with the extra-cellular domain protein of the HER-2 receptor. After each round of selection, a portion of the eluted phagemid were infected into E. coli and plasmid DNA was prepared. This plasmid DNA was then digested with Eco RV and Pst I, separated on a 5% polyacrylamide gel, and stained with ethidium bromide. The bands were visualized under UV light. The bands due to the wild-type and variant plasmids are marked with arrows. The first round of selection was eluted only under acid conditions; subsequent rounds were eluted with either an acid elution (left side of Figure) or with a humanized 4D5 antibody wash step prior to acid elution (right side of Figure) using methods described in Example VIII. Three variant 4D5 Fab molecules were made: H91A (amino acid histidine at position 91 on the VL chain mutated to alanine; indicated as ‘A’ lanes in Figure), Y49A (amino acid tyrosine at position 49 on the VL chain mutated to alanine; indicated as ‘B’ lanes in the Figure), and Y92A (amino acid tyrosine at position 92 on the VL chain mutated to alanine; indicated as ‘C’ lanes in the Figure). Amino acid position numbering is according to Kabat et al., (Sequences of proteins of immunological interest, 4th ed., U.S. Dept of Health and Human Services, Public Health Service, Nat'l. Institute of Health, Bethesda, Md. [1987]). -
FIG. 13 . The Scatchard analysis of the RIA affinity determination described in Experimental Protocols is shown here. The amount of labeled ECD antigen that is bound is shown on the x-axis while the amount that is bound divided by the amount that is free is shown on the y-axis. The slope of the line indicates the Ka; the calculated Kd is 1/Ka. - The following discussion will be best understood by referring to
FIG. 1 . In its simplest form, the method of the instant invention comprises a method for selecting novel binding polypeptides, such as protein ligands, having a desired, usually high, affinity for a target molecule from a library of structurally related binding polypeptides. The library of structurally related polypeptides, fused to a phage coat protein, is produced by mutagenesis and, preferably, a single copy of each related polypeptide is displayed on the surface of a phagemid particle containing DNA encoding that polypeptide. These phagemid particles are then contacted with a target molecule and those particles having the highest affinity for the target are separated from those of lower affinity. The high affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained. - The novel binding polypeptides or ligands produced by the method of this invention are useful per se as diagnostics or therapeutics (eg. agonists or antagonists) used in treatment of biological organisms. Structural analysis of the selected polypeptides may also be used to facilitate rational drug design.
- By “binding polypeptide” as used herein is meant any polypeptide that binds with a selectable affinity to a target molecule. Preferably the polypeptide will be a protein that most preferably contains more than about 100 amino acid residues. Typically the polypeptide will be a hormone or an antibody or a fragment thereof.
- By “high affinity” as used herein is meant an affinity constant (Kd) of <10−5 M and preferably <10−7M under physiological conditions.
- By “target molecule” as used herein is meant any molecule, not necessarily a protein, for which it is desirable to produce a ligand. Preferably, however, the target will be a protein and most preferably the target will be a receptor, such as a hormone receptor.
- By “humanized antibody” as used herein is meant an antibody in which the complementarity-determining regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework. By human antibody framework is meant the entire human antibody excluding the CDRs.
- I. Choice of Polypeptides for Display on the Surface of a Phage
- The first step in the method of this invention is to choose a polypeptide having rigid secondary structure exposed to the surface of the polypeptide for display on the surface of a phage.
- By “polypeptide” as used herein is meant any molecule whose expression can be directed by a specific DNA sequence. The polypeptides of this invention may comprise more than one subunit, where each subunit is encoded by a separate DNA sequence.
- By “rigid secondary structure” as used herein is meant any polypeptide segment exhibiting a regular repeated structure such as is found in; α-helices, 310 helices, π-helices, parallel and antiparallel β-sheets, and reverse turns. Certain “non-ordered” structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or “patch” of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns. The geometry of these rigid secondary structures is well defined by φ and ψ torsional angles about the α-carbons of the peptide “backbone”.
- The requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or “patch” of amino acid residues that can be exposed to and bind with a target molecule. It is primarily these amino acid residues that are replaced by mutagenesis that form the “library” of structurally related (mutant) binding polypeptides that are displayed on the surface of the phage and from which novel polypeptide ligands are selected. Mutagenesis or replacement of amino acid residues directed toward the interior of the polypeptide is generally avoided so that the overall structure of the rigid secondary structure is preserved. Some replacement of amino acids on the interior region of the rigid secondary structures, especially with hydrophobic amino acid residues, may be tolerated since these conservative substitutions are unlikely to distort the overall structure of the polypeptide.
- Repeated cycles of “polypeptide” selection are used to select for higher and higher affinity binding by the phagemid selection of multiple amino acid changes which are selected by multiple selection cycles. Following a first round of phagemid selection, involving a first region or selection of amino acids in the ligand polypeptide, additional rounds of phagemid selection in other regions or amino acids of the ligand polypeptide are conducted. The cycles of phagemid selection are repeated until the desired affinity properties of the ligand polypeptide are achieved. To illustrate this process, Example VIII phagemid selection of hGH was conducted in cycles. In the first cycle
hGH amino acids hGH amino acids hGH amino acids amino acids - From the forgoing it will be appreciated that the amino acid residues that form the binding domain of the polypeptide will not be sequentially linked and may reside on different subunits of the polypeptide. That is, the binding domain tracks with the particular secondary structure at the binding site and not the primary structure. Thus, generally, mutations will be introduced into codons encoding amino acids within a particular secondary structure at sites directed away from the interior of the polypeptide so that they will have the potential to interact with the target. By way of illustration,
FIG. 2 shows the location of residues in hGH that are known to strongly modulate its binding to the hGH-binding protein (Cunningham et al., Science 247:1461-1465 [1990]). Thus representative sites suitable for mutagenesis would includeresidues - There is no requirement that the polypeptide chosen as a ligand to a target normally bind to that target. Thus, for example, a glycoprotein hormone such as TSH can be chosen as a ligand for the FSH receptor and a library of mutant TSH molecules are employed in the method of this invention to produce novel drug candidates.
- This invention thus contemplates any polypeptide that binds to a target molecule, and includes antibodies. Preferred polypeptides are those that have pharmaceutical utility. More preferred polypeptides include; a growth hormone, including human growth hormone, des-N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroid stimulating hormone; thyroxine; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; leutinizing hormone; glucagon; factor VIII; an antibody; lung surfactant; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator (t-PA); bombesin; factor IX, thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as betalactamase; tissue factor protein; inhibin; activin; vascular endothelial growth factor; receptors for hormones or growth factors; integrin; thrombopoietin; protein A or D; rheumatoid factors; nerve growth factor such as NGF-β; platelet-derived growth factor; fibroblast growth factor such as aFGF and bFGF; epidermal growth factor; transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; insulin-like growth factor binding proteins; CD-4; DNase; latency associated peptide; erythropoietin; osteoinductive factors; an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; superoxide dismutase; decay accelerating factor; atrial natriuretic peptides A, B or C; viral antigen such as, for example, a portion of the HIV envelope; immunoglobulins; and fragments of any of the above-listed polypeptides. In addition, one or more predetermined amino acid residues on the polypeptide may be substituted, inserted, or deleted, for example, to produce products with improved biological properties. Further, fragments of these polypeptides, especially biologically active fragments, are included. Yet more preferred polypeptides of this invention are human growth hormone, and atrial naturetic peptides A, B, and C, endotoxin, subtilisin, trypsin and other serine proteases.
- Still more preferred are polypeptide hormones that can be defined as any amino acid sequence produced in a first cell that binds specifically to a receptor on the same cell type (autocrine hormones) or a second cell type (non-autocrine) and causes a physiological response characteristic of the receptor-bearing cell. Among such polypeptide hormones are cytokines, lymphokines, neurotrophic hormones and adenohypophyseal polypeptide hormones such as growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, thyrotropin, chorionic gonadotropin, corticotropin, α or β-melanocyte-stimulating hormone, β-lipotropin, γ-lipotropin and the endorphins; hypothalmic release-inhibiting hormones such as corticotropin-release factor, growth hormone release-inhibiting hormone, growth hormone-release factor; and other polypeptide hormones such as atrial natriuretic peptides A, B or C.
- II. Obtaining a First Gene (Gene 1) Encoding the Desired Polypeptide
- The gene encoding the desired polypeptide (i.e., a polypeptide with a rigid secondary structure) can be obtained by methods known in the art (see generally, Sambrook et al., Molecular Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. [1989]). If the sequence of the gene is known, the DNA encoding the gene may be chemically synthesized (Merrfield, J. Am. Chem. Soc., 85 :2149 [1963]). If the sequence of the gene is not known, or if the gene has not previously been isolated, it may be cloned from a cDNA library (made from RNA obtained from a suitable tissue in which the desired gene is expressed) or from a suitable genomic DNA library. The gene is then isolated using an appropriate probe. For cDNA libraries, suitable probes include monoclonal or polyclonal antibodies (provided that the cDNA library is an expression library), oligonucleotides, and complementary or homologous cDNAs or fragments thereof. The probes that may be used to isolate the gene of interest from genomic DNA libraries include cDNAs or fragments thereof that encode the same or a similar gene, homologous genomic DNAs or DNA fragments, and oligonucleotides. Screening the cDNA or genomic library with the selected probe is conducted using standard procedures as described in chapters 10-12 of Sambrook et al., supra.
- An alternative means to isolating the gene encoding the protein of interest is to use polymerase chain reaction methodology (PCR) as described in
section 14 of Sambrook et al., supra. This method requires the use of oligonucleotides that will hybridize to the gene of interest; thus, at least some of the DNA sequence for this gene must be known in order to generate the oligonucleotides. - After the gene has been isolated, it may be inserted into a suitable vector (preferably a plasmid) for amplification, as described generally in Sambrook et al., supra.
- III. Constructing Replicable Expression Vectors
- While several types of vectors are available and may be used to practice this invention, plasmid vectors are the preferred vectors for use herein, as they may be constructed with relative ease, and can be readily amplified. Plasmid vectors generally contain a variety of components including promoters, signal sequences, phenotypic selection genes, origin of replication sites, and other necessary components as are known to those of ordinary skill in the art.
- Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter, the bacteriophage λPL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter. For general descriptions of promoters, see
section 17 of Sambrook et al. supra. While these are the most commonly used promoters, other suitable microbial promoters may be used as well. - Preferred promoters for practicing this invention are those that can be tightly regulated such that expression of the fusion gene can be controlled. It is believed that the problem that went unrecognized in the prior art was that display of multiple copies of the fusion protein on the surface of the phagemid particle lead to multipoint attachment of the phagemid with the target. It is believed this effect, referred to as the “chelate effect”, results in selection of false “high affinity” polypeptides when multiple copies of the fusion protein are displayed on the phagemid particle in close proximity to one another so that the target was “chelated”. When multipoint attachment occurs, the effective or apparent Kd may be as high as the product of the individual Kds for each copy of the displayed fusion protein. This effect may be the reason Cwirla and coworkers supra were unable to separate moderate affinity peptides from higher affinity peptides.
- It has been discovered that by tightly regulating expression of the fusion protein so that no more than a minor amount, i.e. fewer than about 1%, of the phagemid particles contain multiple copies of the fusion protein the “chelate effect” is overcome allowing proper selection of high affinity polypeptides. Thus, depending on the promoter, culturing conditions of the host are adjusted to maximize the number of phagemid particles containing a single copy of the fusion protein and minimize the number of phagemid particles containing multiple copies of the fusion protein.
- Preferred promoters used to practice this invention are the lac Z promoter and the pho A promoter. The lac Z promoter is regulated by the lac repressor protein lac i, and thus transcription of the fusion gene can be controlled by manipulation of the level of the lac repressor protein. By way of illustration, the phagemid containing the lac Z promotor is grown in a cell strain that contains a copy of the lac i repressor gene, a repressor for the lac Z promotor. Exemplary cell strains containing the lac i gene include JM 101 and XL1-blue. In the alternative, the host cell can be cotransfected with a plasmid containing both the repressor lac i and the Lac Z promotor. Occasionally both of the above techniques are used simultaneously, that is, phagmide particles containing the Lac Z promoter are grown in cell strains containing the lac i gene and the cell strains are cotransfected with a plasmid containing both the lac Z and lac i genes. Normally when one wishes to express a gene, to the transfected host above one would add an inducer such as isopropylthiogalactoside (IPTG). In the present invention however, this step is omitted to (a) minimize the expression of the gene III fusion protein thereby minimizing the copy number (i.e. the number of gene III fusions per phagemid number) and to (b) prevent poor or improper packaging of the phagemid caused by inducers such as IPTG even at low concentrations. Typically, when no inducer is added, the number of fusion proteins per phagemid particle is about 0.1 (number of bulk fusion proteins/number of phagemid particles). The most preferred promoter used to practice this invention is pho A. This promoter is believed to be regulated by the level of inorganic phosphate in the cell where the phosphate acts to down-regulate the activity of the promoter. Thus, by depleting cells of phosphate, the activity of the promoter can be increased. The desired result is achieved by growing cells in a phosphate enriched medium such as 2YT or LB thereby controlling the expression of the gene III fusion.
- One other useful component of vectors used to practice this invention is a signal sequence. This sequence is typically located immediately 5′ to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other 5′ to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell. The DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence. Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF (Wong et al., Gene, 68:193 [1983]), MalE, PhoA and other genes. A preferred prokaryotic signal sequence for practicing this invention is the E. coli heat-stable enterotoxin II (STII) signal sequence as described by Chang et al., Gene, 55: 189 [1987].
- Another useful component of the vectors used to practice this invention is phenotypic selection genes. Typical phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell. By way of illustration, the ampicillin resistance gene (amp), and the tetracycline resistance gene (tet) are readily employed for this purpose.
- Construction of suitable vectors comprising the aforementioned components as well as the gene encoding the desired polypeptide (gene 1) are prepared using standard recombinant DNA procedures as described in Sambrook et al. supra. Isolated DNA fragments to be combined to form the vector are cleaved, tailored, and ligated together in a specific order and orientation to generate the desired vector.
- The DNA is cleaved using the appropriate restriction enzyme or enzymes in a suitable buffer. In general, about 0.2-1 μg of plasmid or DNA fragments is used with about 1-2 units of the appropriate restriction enzyme in about 20 μl of buffer solution. Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the manufacturers of the restriction enzymes. Generally, incubation times of about one or two hours at 37° C. are adequate, although several enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and the DNA is recovered from the aqueous fraction by precipitation with ethanol.
- To ligate the DNA fragments together to form a functional vector, the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the sticky ends commonly produced by endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C. with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation.
- The cleaved DNA fragments may be size-separated and selected using DNA gel electrophoresis. The DNA may be electrophoresed through either an agarose or a polyacrylamide matrix. The selection of the matrix will depend on the size of the DNA fragments to be separated. After electrophoresis, the DNA is extracted from the matrix by electroelution, or, if low-melting agarose has been used as the matrix, by melting the agarose and extracting the DNA from it, as described in sections 6.30-6.33 of Sambrook et al., supra.
- The DNA fragments that are to be ligated together (previously digested with the appropriate restriction enzymes such that the ends of each fragment to be ligated are compatible) are put in solution in about equimolar amounts. The solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 μg of DNA. If the DNA fragment is to be ligated into a vector, the vector is at first linearized by cutting with the appropriate restriction endonuclease(s). The linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. The phosphatasing prevents self-ligation of the vector during the ligation step.
- After ligation, the vector with the foreign gene now inserted is transformed into a suitable host cell. Prokaryotes are the preferred host cells for this invention. Suitable prokaryotic host cells include E. coli strain JM101, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCC number 31,537), E. coli XL-1 Blue (stratagene), and E. coli B; however many other strains of E. coli, such as HB101, NM522, NM538, NM539, and many other species and genera of prokaryotes may be used as well. In addition to the E. coli strains listed above, bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts.
- Transformation of prokaryotic cells is readily accomplished using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation (Neumann et al., EMBO J., 1:841 [1982]) may be used to transform these cells. The transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
- After selection of the transformed cells, these cells are grown in culture and the plasmid DNA (or other vector with the foreign gene inserted) is then isolated. Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale preparation of DNA and the large-scale preparation of DNA as described in sections 1.25-1.33 of Sambrook et al., supra. The isolated DNA can be purified by methods known in the art such as that described in section 1.40 of Sambrook et al., supra. This purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is generally performed by either the method of Messing et al. Nucleic Acids Res., 9:309 [1981] or by the method of Maxam et al. Meth. Enzymol., 65: 499 [1980].
- IV. Gene Fusion
- This invention contemplates fusing the gene enclosing the desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is generated during transcription.
Gene 2 is typically a coat protein gene of a phage, and preferably it is the phage M13 gene III coat protein, or a fragment thereof. Fusion ofgenes gene 2 into a particular site on a plasmid that containsgene 1, or by insertinggene 1 into a particular site on a plasmid that containsgene 2. - Insertion of a gene into a plasmid requires that the plasmid be cut at the precise location that the gene is to be inserted. Thus, there must be a restriction endonuclease site at this location (preferably a unique site such that the plasmid will only be cut at a single location during restriction endonuclease digestion). The plasmid is digested, phosphatased, and purified as described above. The gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be accomplished if the ends of the plasmid are compatible with the ends of the gene to be inserted. If the restriction enzymes are used to cut the plasmid and isolate the gene to be inserted create blunt ends or compatible sticky ends, the DNAs can be ligated together directly using a ligase such as bacteriophage T4 DNA ligase and incubating the mixture at 16° C. for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of Sambrook et al., supra. If the ends are not compatible, they must first be made blunt by using the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both of which require the four deoxyribonucleotide triphosphates to fill-in overhanging single-stranded ends of the digested DNA. Alternatively, the ends may be blunted using a nuclease such as nuclease S1 or mung-bean nuclease, both of which function by cutting back the overhanging single strands of DNA. The DNA is then religated using a ligase as described above. In some cases, it may not be possible to blunt the ends of the gene to be inserted, as the reading frame of the coding region will be altered. To overcome this problem, oligonucleotide linkers may be used. The linkers serve as a bridge to connect the plasmid to the gene to be inserted. These linkers can be made synthetically as double stranded or single stranded DNA using standard methods. The linkers have one end that is compatible with the ends of the gene to be inserted; the linkers are first ligated to this gene using ligation methods described above. The other end of the linkers is designed to be compatible with the plasmid for ligation. In designing the linkers, care must be taken to not destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid. In some cases, it may be necessary to design the linkers such that they code for part of an amino acid, or such that they code for one or more amino acids.
- Between
gene 1 andgene 2, DNA encoding a termination codon may be inserted, such termination codons are UAG (amber), UM (ocher) and UGA (opel). (Microbiology, Davis et al. Harper & Row, New York, 1980,pages 237, 245-47 and 274). The termination codon expressed in a wild type host cell results in the synthesis of thegene 1 protein product without thegene 2 protein attached. However, growth in a suppressor host cell results in the synthesis of detectable quantities of fused protein. Such suppressor host cells contain a tRNA modified to insert an amino acid in the termination codon position of the mRNA thereby resulting in production of detectible amounts of the fusion protein. Such suppressor host cells are well known and described, such as E. coli suppressor strain (Bullock et al.,BioTechniques 5, 376-379 [1987]). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide. - The suppressible codon may be inserted between the first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein. Alternatively, the suppressible termination codon may be inserted adjacent to the fusion site by replacing the last amino acid triplet in the polypeptide or the first amino acid in the phage coat protein. When the phagemid containing the suppressible codon is grown in a suppressor host cell, it results in the detectable production of a fusion polypeptide containing the polypeptide and the coat protein. When the phagemid is grown in a non-suppressor host cell, the polypeptide is synthesized substantially without fusion to the phage coat protein due to termination at the inserted suppressible triplet encoding UAG, UAA, or UGA. In the non-suppressor cell the polypeptide is synthesized and secreted from the host cell due to the absence of the fused phage coat protein which otherwise anchored it to the host cell.
- V. Alteration (Mutation) of
Gene 1 at Selected Positions -
Gene 1, encoding the desired polypeptide, may be altered at one or more selected codons. An alteration is defined as a substitution, deletion, or insertion of one or more codons in the gene encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the unaltered or native sequence of the same polypeptide. Preferably, the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule. The alterations may be produced be a variety of methods known in the art. These methods include but are not limited to oligonucleotide-mediated mutagenesis and cassette mutagenesis. - A. Oligonucleotide-Mediated Mutagenesis
- Oligonucleotide-mediated mutagenesis is preferred method for preparing substitution, deletion, and insertion variants of
gene 1. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 [1987]. Briefly,gene 1 is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single-stranded form of the plasmid containing the unaltered or native DNA sequence ofgene 1. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template will thus incorporate the oligonucleotide primer, and will code for the selected alteration ingene 1. - Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. Proc. Nat'l. Acad. Sci. USA, 75: 5765 [1978].
- The DNA template can only be generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13 mp18 and M13 mp19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al. Meth. Enzymol., 153: 3 [1987]. Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.
- To alter the native DNA sequence, the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. A heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of
gene 1, and the other strand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. Coli JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA. - The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single-stranded oligonucleotide is annealed to the single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell such as E. coli JM101, as described above.
- Mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
- In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. The alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.
- B. Cassette Mutagenesis
- This method is also a preferred method for preparing substitution, deletion, and insertion variants of
gene 1. The method is based on that described by Wells et al. Gene, 34:315 [1985]. The starting material is the plasmid (or other vector) comprisinggene 1, the gene to be mutated. The codon(s) ingene 1 to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations ingene 1. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3′ and 5′ ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence ofgene 1. - VI. Obtaining DNA Encoding the Desired Protein
- In an alternative embodiment, this invention contemplates production of variants of a desired protein containing one or more subunits. Each subunit is typically encoded by separate gene. Each gene encoding each subunit can be obtained by methods known in the art (see, for example, Section II). In some instances, it may be necessary to obtain the gene encoding the various subunits using separate techniques selected from any of the methods described in Section II.
- When constructing a replicable expression vector where the protein of interest contains more than one subunit, all subunits can be regulated by the same promoter, typically located 5′ to the DNA encoding the subunits, or each may be regulated by separate promoter suitably oriented in the vector so that each promoter is operably linked to the DNA it is intended to regulate. Selection of promoters is carried out as described in Section III above.
- In constructing a replicable expression vector containing DNA encoding the protein of interest having multiple subunits, the reader is referred to
FIG. 10 where, by way of illustration, a vector is diagrammed showing DNA encoding each subunit of an antibody fragment. This figure shows that, generally, one of the subunits of the protein of interest will be fused to a phage coat protein such as M13 gene III. This gene fusion generally will contain its own signal sequence. A separate gene encodes the other subunit or subunits, and it is apparent that each subunit generally has its own signal sequence.FIG. 10 also shows that a single promoter can regulate the expression of both subunits. Alternatively, each subunit may be independently regulated by a different promoter. The protein of interest subunit-phage coat protein fusion construct can be made as described in Section IV above. - When constructing a family of variants of the desired multi-subunit protein, DNA encoding each subunit in the vector may mutated in one or more positions in each subunit. When multi-subunit antibody variants are constructed, preferred sites of mutagenesis correspond to codons encoding amino acid residues located in the complementarity-determining regions (CDR) of either the light chain, the heavy chain, or both chains. The CDRs are commonly referred to as the hypervariable regions. Methods for mutagenizing DNA encoding each subunit of the protein of interest are conducted essentially as described in Section V above.
- VII. Preparing a Target Molecule and Binding with Phagemid
- Target proteins, such as receptors, may be isolated from natural sources or prepared by recombinant methods by procedures known in the art. By way of illustration, glycoprotein hormone receptors may be prepared by the technique described by McFarland et al., Science 245:494-499 [1989], nonglycosylated forms expressed in E. coli are described by Fuh et al. J. Biol. Chem. 265:3111-3115 [1990] Other receptors can be prepared by standard methods.
- The purified target protein may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxylalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44 [1976], or by other means known in the art.
- After attachment of the target protein to the matrix, the immobilized target is contacted with the library of phagemid particles under conditions suitable for binding of at least a portion of the phagemid particles with the immobilized target. Normally, the conditions, including pH, ionic strength, temperature and the like will mimic physiological conditions.
- Bound phagemid particles (“binders”) having high affinity for the immobilized target are separated from those having a low affinity (and thus do not bind to the target) by washing. Binders may be dissociated from the immobilized target by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
- Suitable host cells are infected with the binders and helper phage, and the host cells are cultured under conditions suitable for amplification of the phagemid particles. The phagemid particles are then collected and the selection process is repeated one or more times until binders having the desired affinity for the target molecule are selected.
- Optionally the library of phagemid particles may be sequentially contacted with more than one immobilized target to improve selectivity for a particular target. For example, it is often the case that a ligand such as hGH has more than one natural receptor. In the case of hGH, both the growth hormone receptor and the prolactin receptor bind the hGH ligand. It may be desirable to improve the selectivity of hGH for the growth hormone receptor over the prolactin receptor. This can be achieved by first contacting the library of phagemid particles with immobilized prolactin receptor, eluting those with a low affinity (i.e. lower than wild type hGH) for the prolactin receptor and then contacting the low affinity prolactin “binders” or non-binders with the immobilized growth hormone receptor, and selecting for high affinity growth hormone receptor binders. In this case an hGH mutant having a lower affinity for the prolactin receptor would have therapeutic utility even if the affinity for the growth hormone receptor were somewhat lower than that of wild type hGH. This same strategy may be employed to improve selectivity of a particular hormone or protein for its primary function receptor over its clearance receptor.
- In another embodiment of this invention, an improved substrate amino acid sequence can be obtained. These may be useful for making better “cut sites” for protein linkers, or for better protease substrates/inhibitors. In this embodiment, an immobilizable molecule (e.g. hGH-receptor, biotin-avidin, or one capable of covalent linkage with a matrix) is fused to gene III through a linker. The linker will preferably be from 3 to 10 amino acids in length and will act as a substrate for a protease. A phagemid will be constructed as described above where the DNA encoding the linker region is randomly mutated to produce a randomized library of phagemid particles with different amino acid sequences at the linking site. The library of phagemid particles are then immobilized on a matrix and exposed to a desired protease. Phagemid particles having preferred or better substrate amino acid sequences in the liner region for the desired protease will be eluted, first producing an enriched pool of phagemid particles encoding preferred linkers. These phagemid particles are then cycled several more times to produce an enriched pool of particles encoding consense sequence(s) (see examples XIII and XIV).
- VIII. Growth Hormone Variants and Methods of Use
- The cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N>, et al., [1987]
Gene 55 189) and its DNA and amino acid sequence has been reported (Goeddel, et al. [1979] Nature 281, 544; Gray et al., [1985] Gene 39, 247). The present invention describes novel hGH variants produced using the phagemid selection methods. Human growth hormone variants containing substitutions atpositions - Therapeutic formulations of hGH for therapeutic administration are prepared for storage by mixing hGH having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980)., 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; divalent metal ions such as zinc, cobalt or copper; 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). Formulations of the present invention may additionally contain a pharmaceutically acceptable buffer, amino acid, bulking agent and/or non-ionic surfactant. These include, for example, buffers, chelating agents, antioxidants, preservatives, cosolvents, and the like; specific examples of these could include, trimethylamaine salts (“Tris buffer”), and disodium edetate. The phagemids of the present invention may be used to produce quantities of the hGH variants free of the phage protein. To express hGH variants free of the gene III portion of the fusion, pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction. The hGH variant is secreted from the host and may be isolated from the culture medium.
- One or more of the eight hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I 179 may be replaced by any amino acid other than the one found in that position in naturally occurring hGH as indicated. Therefore, 1, 2, 3, 4, 5, 6, 7, or all 8 of the indicated amino acids, F10, M14, H18, H21, R167, D171, T175 and I 179, may be replaced by any of the other 19 amino acids out of the 20 amino acids listed below. In a preferred embodiment, all eight listed amino acids are replaced by another amino acid. The most preferred eight amino acids to be substituted are indicated in Table XIV in Example XII.
Amino acid nomenclature. Ala (A) Arg (R) Asn (N) Asp (D) Cys (C) Gln (Q) Glu (E) Gly (G) His (H) Ile (I) Leu (L) Lys (K) Met (M) Phe (F) Pro (P) Ser (S) Thr (T) Trp (W) Tyr (Y) Val (V)
The one letter hGH variant nomenclature first gives the hGH amino acid deleted, forexample glutamate 179; then the amino acid inserted; for example, serine; resulting in (E1795S). - Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and illustrative examples, make and utilize the present invention to the fullest extent. The following working examples therefore specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way of the remainder of the disclosure.
- The plasmid phGH-M13gIII (
FIG. 1 ), was constructed from M13KO77 and the hGH producing plasmid, pBO473 (Cunningham, B. C., et al., Science, 243:1330-1336, [1989]). Asynthetic oligonucleotide 5′-AGC-TGT-GGC-TTC-GGG-CCC-TTA-GCA-TTT-AAT-GCG-GTA-3′ (SEQ ID NO:2) was used to introduce a unique ApaI restriction site (underlined) into pBO473 after the final Phe191 codon of hGH. Theoligonucleotide 5′-TTC-ACA-AAC-GAA-GGG-CCC-CTA-ATT-AAA-GCC-AGA-3′ (SEQ ID NO:3) was used to introduce a unique ApaI restriction site (underlined), and a Glu197-to-amber stop codon (hold lettering) into M13KO7 gene III. Theoligonucleotide 5′-CAA-TAA-TAA-CGG-GCT-AGC-CAA-AAG-AAC-TGG-3′ (SEQ ID NO:4) introduces a unique NheI site (underlined) after the 3′ end of the gene III coding sequence. The resulting 650 base pair (bp) ApaI-NheI fragment from the doubly mutated M13KO7 gene III was cloned into the large ApaI-NheI fragment of pBO473 to create the plasmid, pSO132. This fuses the carboxyl terminus of hGH (Phe191) to the Pro198 residue of the gene III protein with the insertion of a glycine residue encoded from the ApaI site and places the fusion protein under control of the E. coli alkaline phosphatase (phoA) promoter and stII secretion signal sequence (Chang, C. N., et al, Gene, 55:189-196, [1987]). For inducible expression of the fusion protein in rich media, we replaced the phoA promoter with the lac promoter and operator. A 138 bp EcoRI-XbaI fragment containing the lac promoter, operator, and Cap binding site was produced by PCR of plasmid pUC119 using theoligonucleotides 5′-CACGACAGAATTCCCGACTGGAAA-3′ (SEQ ID NO:5) and 5′-CTGTT TCTAGAGTGAAATTGTTA-3′ (SEQ ID NO:6) that flank the desired lac sequences and introduce the EcoRI and XbaI restriction sites (underlined). This lac fragment was gel purified and ligated into the large EcoRI-XbaI fragment of pSO132 to create the plasmid, phGH-M13gIII. The sequences of all tailored DNA junctions were verified by the dideoxy sequence method (Sanger, F., et al. Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467, [1977]). The R64A variant hGH phagemid was constructed as follows: the NsiI-BglII mutated fragment of hGH (Cunningham et al. supra) encoding the Arg64 to Ala substitution (R64A) (Cunningham, B. C., Wells, J. A., Science, 244:1081-1085, [1989]) was cloned between the corresponding restriction sites in the phGH-M13gIII plasmid (FIG. 1 ) to replace the wild-type hGH sequence. The R64A hGH phagemid particles were propagated and titered as described below for the wild-type hGH-phagemid. - Plasmids were transformed into a male strain of E. coli (JM101) and selected on carbenicillin plates. A single transformant was grown in 2 ml 2YT medium for 4 h at 37° C. and infected with 50 μl of M13KO7 helper phage. The infected culture was diluted into 30 ml 2YT, grown overnight, and phagemid particles were harvested by precipitation with polyethylene glycol (Vierra, J., Messing, J., Methods in Enzymology, 153:3-11, [1987]). Typical phagemid particle titers ranged from 2 to 5×1011 cfu/ml. The particles were purified to homogeneity by CsCl density centrifugation (Day, L. A.J. Mol. Biol., 39:265-277, [1969]) to remove any fusion protein not attached to virions.
- Rabbit polyclonal antibodies to hGH were purified with protein A, and coated onto microtiter plates (Nunc) at a concentration of 2 μg/ml in 50 mM sodium carbonate buffer (pH 10) at 4° C. for 16-20 hours. After washing in PBS containing 0.05
% Tween 20, hGH or hGH-phagemid particles were serially diluted from 2.0-0.002 nM in buffer A (50 mM Tris (pH 7.5), 50 mM NaCl, 2 mM EDTA, 5 mg/ml bovine serum albumin, and 0.05% Tween 20). After 2 hours at room temperature (rt), the plates were washed well and the indicated Mab (Cunningham et al. supra) was added at 1 μg/ml in buffer A for 2 hours at rt. Following washing, horseradish peroxidase conjugated goat anti-mouse IgG antibody was bound at rt for 1 hour. After a final wash, the peroxidase activity was assayed with the substrate, o-phenylenediamine. - Oxirane polyacrylamide beads (Sigma) were conjugated to the purified extracellular domain of the hGH receptor (hGHbp) (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 [1990]) containing an extra cysteine residue introduced by site-directed mutagenesis at
position 237 that does not affect binding of hGH (J. Wells, unpublished). The hGHbp was conjugated as recommended by the supplier to a level of 1.7 μmol hGHbp/mg dry oxirane bead, as measured by binding of [125I] hGH to the resin. Subsequently, any unreacted oxirane groups were blocked with BSA and Tris. As a control for non-specific binding of phagemid particles, BSA was similarly coupled to the beads. Buffer for adsorption and washing contained 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg/ml BSA, and 0.02% Tween 20. Elution buffers contained wash buffer plus 200 nM hGH or 0.2 M glycine (pH 2.1). Parental phage M13KO7 was mixed with hGH phagemid particles at a ratio of nearly 3000:1 (original mixture) and tumbled for 8-12 h with a 5 μl aliquot (0.2 mg of acrylamide beads) of either absorbent in a 50 μl volume at room temperature. The beads were pelleted by centrifugation and the supernate carefully removed. The beads were resuspended in 200 μl wash buffer and tumbled at room temperature for 4 hours (wash 1). After a second wash (wash 2), the beads were eluted twice with 200 nM hGH for 6-10 hours each (eluate 1, eluate 2). The final elution was with a glycine buffer (pH 2.1) for 4 hours to remove remaining hGH phagemid particles (eluate 3). Each fraction was diluted appropriately in 2YT media, mixed with fresh JM101, incubated at 37° C. for 5 minutes, and plated with 3 ml of 2YT soft agar on LB or LB carbenicillin plates. - The gene III protein is composed of 410 residues divided into two domains that are separated by a flexible linker sequence (Armstrong, J., et al., FEBS Lett., 135:167-172, [1981]). The amino-terminal domain is required for attachment to the pili of E. coli, while the carboxyl-terminal domain is imbedded in the phage coat and required for proper phage assembly (Crissman, J. W., Smith, G. P., Virology, 132:445-455, [1984]). The signal sequence and amino-terminal domain of gene III was replaced with the stII signal and entire hGH gene (Chang et al. supra) by fusion to residue 198 in the carboxyl-terminal domain of gene III (
FIG. 1 ). The hGH-gene III fusion was placed under control of the lac promoter/operator in a plasmid (phGH-M13gIII;FIG. 1 ) containing the pBR322 β-lactamase gene and Col E1 replication origin, and the phage f1 intergenic region. The vector can be easily maintained as a small plasmid vector by selection on carbenicillin, which avoids relying on a functional gene III fusion for propagation. Alternatively, the plasmid can be efficiently packaged into virions (called phagemid particles) by infection with helper phage such as M13KO7 (Viera et al. supra) which avoids problems of phage assembly. Phagemid infectivity titers based upon transduction to carbenicillin resistance in this system varied from 2-5×1011 colony forming units (cfu)/ml. The titer of the M13KO7 helper phage in these phagemid stocks is ˜1010 plaque forming units (pfu)/ml. - With this system we confirmed previous studies (Parmley, Smith supra) that homogeneous expression of large proteins fused to gene III is deleterious to phage production (data not shown). For example, induction of the lac promoter in phGH-M13gIII by addition of IPTG produced low phagemid titers. Moreover, phagemid particles produced by co-infection with M13KO7 containing an amber mutation in gene III gave very low phagemid titers (<1010 cfu/ml). We believed that multiple copies of the gene III fusion attached to the phagemid surface could lead to multiple point attachment (the “chelate effect”) of the fusion phage to the immobilized target protein. Therefore to control the fusion protein copy number we limited transcription of the hGH-gene III fusion by culturing the plasmid in E. coli JM101 (lacIQ) which contains a constitutively high level of the lac repressor protein. The E. coli JM101 cultures containing phGH-M13gIII were best propagated and infected with M13KO7 in the absence of the lac operon inducer (IPTG); however, this system is flexible so that co-expression of other gene III fusion proteins can be balanced. We estimate that about 10% of the phagemid particles contain one copy of the hGH gene III fusion protein from the ratio of the amount of hGH per virion (based on hGH immuno-reactive material in CsCl gradient purified phagemid). Therefore, the titer of fusion phage displaying the hGH gene III fusion is about 2−5×1010/ml. This number is much greater than the titer of E. coli (˜108 to 109/ml) in the culture from which they are derived. Thus, on average every E. coli cell produces 10-100 copies of phage decorated with an hGH gene III fusion protein.
- Immunoblot analysis (
FIG. 2 ) of the hGH-gene III phagemid show that hGH cross-reactive material comigrates with phagemid particles in agarose gels. This indicates that the hGH is tightly associated with phagemid particles. The hGH-gene III fusion protein from the phagemid particles runs as a single immuno-stained band showing that there is little degradation of the hGH when it is attached to gene III. Wild-type gene III protein is clearly present because about 25% of the phagemid particles are infectious. This is comparable to specific infectivity estimates made for wild-type M13 phage that are similarly purified (by CsCl density gradients) and concentrations estimated by UV absorbance (Smith, G. P. supra and Parmley, Smith supra) Thus, both wild-type gene III and the hGH-gene III fusion proteins are displayed in the phage pool. - It was important to confirm that the tertiary structure of the displayed hGH was maintained in order to have confidence that results from binding selections will translate to the native protein. We used monoclonal antibodies (Mabs) to hGH to evaluate the structural integrity of the displayed hGH gene III fusion protein (Table I).
TABLE I Binding of Eight Different Monoclonal Antibodies (Mab's) to hGH and hGH Phagemid Particles* IC50 (nM) Mab hGH hGH- phagemid 1 0.4 0.4 2 0.04 0.04 3 0.2 0.2 4 0.1 0.1 5 0.2 >2.0 6 0.07 0.2 7 0.1 0.1 8 0.1 0.1
*Values given represent the concentration (nM) of hGH or hGH-phagemid particles to give half-maximal binding to the particular Mab. Standard errors in these measurements are typically at or below ±30% of the reported value. See Materials and Methods for further details.
- The epitopes on hGH for these Mabs have been mapped (Cunningham et al. supra) and binding for 7 of 8 Mabs requires that hGH be properly folded. The IC50 values for all Mabs were equivalent to wild-type hGH except for
Mab Mabs - Previous workers (Parmley, Smith supra; Scott, Smith supra; Cwirla et al. supra; and Devlin et al. supra) have fractionated phage by panning with streptavidin coated polystyrene petri dishes or microtiter plates. However, chromatographic systems would allow more efficient fractionation of phagemid particles displaying mutant proteins with different binding affinities. We chose non-porous oxirane beads (Sigma) to avoid trapping of phagemid particles in the chromatographic resin. Furthermore, these beads have a small particle size (1 μm) to maximize the surface area to mass ratio. The extracellular domain of the hGH receptor (hGHbp) (Fuh et al., supra) containing a free cysteino residue was efficiently coupled to these beads and phagemid particles showed very low non-specific binding to beads coupled only to bovine serum albumin (Table II).
TABLE II Specific Binding of Hormone Phage to hGHbp-coated Beads Provides an Enrichment for hGH-phage over M13KO7 Phage* Sample Absorbent‡ Total pfu Total cfu Ratio (cfu/pfu) Enrichment§ Original mixture† 8.3 × 1011 2.9 × 108 3.5 × 10−4 (1) Supernatant BSA 7.4 × 1011 2.8 × 108 3.8 × 10−4 1.1 hGHbp 7.6 × 1011 3.3 × 108 4.3 × 10−4 1.2 Wash 1BSA 1.1 × 1010 6.0 × 106 5.5 × 10−4 1.6 hGHbp 1.9 × 1010 1.7 × 107 8.9 × 10−4 2.5 Wash 2BSA 5.9 × 107 2.8 × 104 4.7 × 10−4 1.3 hGHbp 4.9 × 107 2.7 × 106 5.5 × 10−2 1.6 × 102 Eluate 1 (hGH) BSA 1.1 × 106 1.9 × 103 1.7 × 10−3 4.9 hGHbp 1.2 × 106 2.1 × 106 1.8 5.1 × 103 Eluate 2 (hGH) BSA 5.9 × 105 1.2 × 103 2.0 × 10−3 5.7 hGHbp 5.5 × 105 1.3 × 106 2.4 6.9 × 103 Eluate 3 (pH 2.1) BSA 4.6 × 105 2.0 × 103 4.3 × 10−3 12.3 hGHbp 3.8 × 105 4.0 × 106 10.5 3.0 × 104
*The titers of M13KO7 and hGH-phagemid particles in each fraction was determined by multiplying the number of plaque forming units (pfu) or carbenicillin resistant colony forming units (cfu) by the dilution factor, respectively. See Example IV for details.
†The ratio of M13KO7 to hGH-phagemid particles was adjusted to 3000:1 in the original mixture.
‡Absorbents were conjugated with BSA or hGHbp.
§Enrichments are calculated by dividing the cfu/pfu ratio after each step by cfu/pfu ratio in the original mixture.
- In a typical enrichment experiment (Table II), one part of hGH phagemid was mixed with >3,000 parts M13KO7 phage. After one cycle of binding and elution, 106 phage were recovered and the ratio of phagemid to M13KO7 phage was 2 to 1. Thus, a single binding selection step gave >5000-fold enrichment. Additional elutions with free hGH or acid treatment to remove remaining phagemids produced even greater enrichments. The enrichments are comparable to those obtained by Smith and coworkers using batch elution from coated polystyrene plates (Smith, G. P. supra and Parmely, Smith supra) however much smaller volumes are used on the beads (200 μl vs. 6 ml). There was almost no enrichment for the hGH phagemid over M13KO7 when we used beads linked only to BSA. The slight enrichment observed for control beads (˜10-fold for pH 2.1 elution; Table 2) may result from trace contaminants of bovine growth hormone binding protein present in the BSA linked to the bead. Nevertheless these data show the enrichments for the hGH phage depend upon the presence of the hGHbp on the bead suggesting binding occurs by specific interaction between hGH and the hGHbp.
- We evaluated the enrichment for wild-type hGH over a weaker binding variant of the hGH on fusion phagemids to further demonstrate enrichment specificity, and to link the reduction in binding affinity for the purified hormones to enrichment factors after panning fusion phagemids. A fusion phagemid was constructed with an hGH mutant in which Arg64 was substituted with Ala (R64A). The R64A variant hormone is about 20-fold reduced in receptor binding affinity compared to hGH (Kd values of 7.1 nM and 0.34 nM, respectively [Cunningham, Wells, supra]). The titers of the R64A hGH-gene III fusion phagemid were comparable to those of wild-type hGH phagemid. After one round of binding and elution (Table III) the wild-type hGH phagemid was enriched from a mixture of the two phagemids plus M13KO7 by 8-fold relative to the phagemid R64A, and ˜104 relative to M13KO7 helper phage.
TABLE III hGHbp-coated Beads Select for hGH Phagemids Over a Weaker Binding hGH Variant Phagemid Control beads hGHbp beads WT phagemid enrichment WT phagemid enrichment Sample total phagemid for WT/R64A total phagemid for WT/ R64A Original mixture 8/20 (1) 8/20 (1) Supernatant ND — 4/10 1.0 Elution 1 (hGH) 7/20 0.8 17/20 8.5‡ Elution 2 (pH 2.1) 11/20 1.8 21/27 5.2
* The parent M13KO7 phage, wild-type hGH phagemid and R64A phagemid particles were mixed at a ratio of 104:0.4:0.6. Binding selections were carried out using beads linked with BSA (control beads) or with the hGHbp (hGHbp beads) as described in Table II and the Materials and Methods After each step, plasmid DNA was isolated (Birnboim, H. C., Doly, J., Nucleic Acids Res., 7: 1513-1523, [1979]) from carbenicillin resistant colonies and analyzed by restriction analysis
# to determine if it contained the wild-type hGH or the R64A hGH gene III fusion.
† The enrichment for wild-type hGH phagemid over R64A mutant was calculated from the ratio of hGH phagemid present after each step to that present in the original mixture (8/20), divided by the corresponding ratio for R64A phagemids. WT = wild-type; ND = not determined.
‡The enrichment for phagemid over total M13KO7 parental phage was ˜104 after this step.
- By displaying a mixture of wild-type gene III and the gene III fusion protein on phagemid particles one can assemble and propagate virions that display a large and proper folded protein as a fusion to gene III. The copy number of the gene III fusion protein can be effectively controlled to avoid “chelate effects” yet maintained at high enough levels in the phagemid pool to permit panning of large epitope libraries (>1010). We have shown that hGH (a 22 kD protein) can be displayed in its native folded form. Binding selections performed on receptor affinity beads eluted with free hGH, efficiently enriched for wild-type hGH phagemids over a mutant hGH phagemid shown to have reduced receptor binding affinity. Thus, it is possible to sort phagemid particles whose binding constants are down in the nanomolar range.
- Protein-protein and antibody-antigen interactions are dominated by discontinuous epitopes (Janin, J., et al., J. Mol. Biol., 204:155-164, [1988]; Argos, P., Prot. Eng., 2:101-113, [1988]; Barlow, D. J., et al., Nature, 322:747-748, [1987]; add Davies, D. R., et al., J. Biol. Chem., 263:10541-10544, [1988]); that is the residues directly involved in binding are close in tertiary structure but separated by residues not involved in binding. The screening system presented here should allow one to analyze more conveniently protein-receptor interactions and isolate discontinuous epitopes in proteins with new and high affinity binding properties.
- Construction of Template
- A mutant of the hGH-gene III fusion protein was constructed using the method of Kunkel., et al. Meth. Enzymol. 154, 367-382 [1987]. Template DNA was prepared by growing the plasmid pS0132 (containing the natural hGH gene fused to the carboxy-terminal half of M13 gene III, under control of the alkaline phosphatase promoter) in CJ236 cells with M13-K07 phage added as helper. Single-stranded, uracil-containing DNA was prepared for mutagenesis to introduce (1) a mutation in hGH which would greatly reduce binding to the hGH binding protein (hGHbp); and (2) a unique restriction site (KpnI) which could be used for assaying for—and selecting against—parental background phage. Oligonucleotide-directed mutagenesis was carried out using T7 DNA polymerase and the following oligodeoxy-nucleotide (SEQ ID NO:7):
Gly Thr hGH codon: 178 179 5′-G ACA TTC CTG GGT ACC GTG CAG T-3′ < KpnI >
This oligo introduces the KpnI site as shown, along with mutations (R178G, I179T) in hGH. These mutations are predicted to reduce binding of hGH to hGHbp by more than 30-fold. Clones from the mutagenesis were screened by KpnI digestion and confirmed by dideoxy DNA sequencing. The resulting construct, to be used as a template for random mutagenesis, was designated pHO415.
Random Mutagenesis within Helix-4 of hGH -
Codons hGH codon: 172 174 5′- GC TTC AGG AAG GAC ATG GAC NNS GTC NNS ACA- Ile 176 178 179 - NNS CTG NNS ATC GTG CAG TGC CGC TCT GTG G-3′
As shown, this oligo pool revertscodon 179 to wild-type (Ile), destroys the unique KpnI site of pH0415, and introduces random codons (NNS, where N=A, G, C, or T and S=G or C) atpositions
Propagation of the Initial Library - The mutagenesis products were extracted twice with phenol:chloroform (50:50) and ethanol precipitated with an excess of carrier tRNA to avoid adding salt that would confound the subsequent electroporation step. Approximately 50 ng (15 fmols) of DNA was electroporated into WJM101 cells (2.8×1010 cells/mL) in 45 μL total volume in a 0.2 cm cuvette at a voltage setting of 2.49 kV with a single pulse (time constant=4.7 msec.).
- The cells were allowed to recover 1 hour at 37° C. with shaking, then mixed with 25 mL 2YT medium, 100 μg/mL carbenicillin, and M13-K07 (multiplicity of infection=1000). Plating of serial dilutions from this culture onto carbenicillin-containing media indicated that 8.2×106 electrotransformants were obtained. After 10′ at 23° C., the culture was incubated overnight (15 hours) at 37° C. with shaking.
- After overnight incubation, the cells were pelleted, and double-stranded DNA (dsDNA), designated pLIB1, was prepared by the alkaline lysis method. The supernatant was spun again to remove any remaining cells, and the phage, designated phage pool φ1, were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p gene III fusion (hGH-g3) plasmid, and plaque-forming units (PFU) for the M13-K07 helper phage.
- Binding Selection Using Immobilized hGHbp
- 1. BINDING: An aliquot of phage pool φ1 (6×109 CFU, 6×107 PFU) was diluted 4.5-fold in buffer A (Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20, 0.01% thimerosal) and mixed with a 5 μL suspension of oxirane-polyacrylamide beads coupled to the hGHbp containing a Ser237 Cys mutation (350 fmols) in a 1.5 mL silated polypropylene tube. As a control, an equivalent aliquot of phage were mixed in a separate tube with beads that had been coated with BSA only. The phage were allowed to bind to the beads by incubating 3 hours at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 μL and at room temperature.
- 2. WASH: The beads were spun 15 sec., and the supernatant was removed (Sup. 1). To remove phage/phagemid not specifically bound, the beads were washed twice by resuspending in buffer A, then pelleting. A final wash consisted of rotating the beads in buffer A for 2 hours.
- 3. hGH ELUTION: Phage/phagemid binding weakly to the beads were removed by stepwise elution with hGH. In the first step, the beads were rotated with buffer A containing 2 nM hGH. After 17 hours, the beads were pelleted and resuspended in buffer A containing 20 nM hGH and rotated for 3 hours, then pelleted. In the final hGH wash, the beads were suspended in buffer A containing 200 nM hGH and rotated for 3 hours then pelleted.
- 4. GLYCINE ELUTION: To remove the tightest-binding phagemid (i.e. those still bound after the hGH washes), beads were suspended in Glycine buffer (1 M Glycine, pH 2.0 with HCl), rotated 2 hours and pelleted. The supernatant (fraction “G”; 200 μL) was neutralized by adding 30 μL of 1 M Tris base.
- Fraction G eluted from the hGHbp-beads (1×106 CFU, 5×104 PFU) was not substantially enriched for phagemid over K07 helper phage. We believe this resulted from the fact that K07 phage packaged during propagation of the recombinant phagemid display the hGH-g3p fusion.
- However, when compared with fraction G eluted from the BSA-coated control beads, the hGHbp-beads yielded 14 times as many CFU's. This reflects the enrichment of tight-binding hGH-displaying phagemid over nonspecifically-binding phagemid.
- 5. PROPAGATION: An aliquot (4.3×105 CFU) of fraction G eluted from the hGHbp-beads was used to infect log-phase WJM101 cells. Transductions were carried out by mixing 100 μL fraction G with 1 mL WJM101 cells, incubating 20 min. at 37° C., then adding K07 (multiplicity of infection=1000). Cultures (25 mL 2YT plus carbenicillin) were grown as described above and the second pool of phage (
Library 1G, for first glycine elution) were prepared as described above. - Phage from
library 1G (FIG. 3 ) were selected for binding to hGHbp beads as described above. Fraction G eluted from hGHbp beads contained 30 times as many CFU's as fraction G eluted from BSA-beads in this selection. Again, an aliquot of fraction G was propagated in WJM101 cells to yieldlibrary 1G2 (indicating that this library had been twice selected by glycine elution). Double-stranded DNA (pLIB 1G2) was also prepared from this culture. - KpnI Assay and Restriction-Selection of dsDNA
- To reduce the level of background (KpnI+) template, an aliquot (about 0.5 μg) of
pLIB 1G2 was digested with KpnI and electroporated into WJM101 cells. These cells were grown in the presence of K07 (multiplicity of infection=100) as described for the initial library, and a new phage pool,pLIB 3, was prepared (FIG. 3 ). - In addition, an aliquot (about 0.5 μg) of dsDNA from the initial library (pLIB1) was digested with KpnI and electroporated directly into WJM101 cells. Transformants were allowed to recover as above, infected with M13-K07, and grown overnight to obtain a new library of phage, designated phage Library 2 (
FIG. 3 ). - Successive Rounds of Selection
- Phagemid binding, elution, and propagation were carried out in successive rounds for phagemid derived from both
pLIB 2 and pLIB 3 (FIG. 3 ) as described above, except that (1) an excess (10-fold over CFU) of purified K07 phage (not displaying hGH) was added in the bead-binding cocktail, and (2) the hGH stepwise elutions were replaced with brief washings of buffer A alone. Also, in some cases, XL1-Blue cells were used for phagemid propagation. - An additional digestion of dsDNA with KpnI was carried out on
pLIB 2G3 and onpLIB 3G5 before the final round of bead-binding selection (FIG. 3 ). - DNA Sequencing of Selected Phagemids
- Four independently isolated clones from
LIB 4G4 and four independently isolated clones fromLIB 5G6 were sequenced by dideoxy sequencing. All eight of these clones had identical DNA sequences (SEQ ID NO:9):hGH codon: 172 174 176 178 5′-AAG GTC TCC ACA TAC CTG AGG ATC-3′
Thus, all these encode the same mutant of hGH: (E174S, F176Y).Residue 172 in these clones is Lys as in wild-type. The codon selected for 172 is also identical to wild-type hGH. This is not surprising since MG is the only lysine-codon possible from a degenerate “NNS” codon set. Residue 178-Arg is also the same as wild-type, but here, the codon selected from the library was MG instead of CGC as is found in wild-type hGH, even though the latter codon is also possible using the “NNS” codon set.
Multiplicity of K07 Infection - The multiplicity of infection of K07 infection is an important parameter in the propagation of recombinant phagemids. The K07 multiplicity of infection must be high enough to insure that virtually all cells transformed or transfected with phagemid are able to package new phagemid particles. Furthermore, the concentration of wild-type gene III in each cell should be kept high to reduce the possibility of multiple hGH-gene III fusion molecules being displayed on each phagemid particle, thereby reducing chelate effects in binding. However, if the K07 multiplicity of infection is too high, the packaging of K07 will compete with that of recombinant phagemid. We find that acceptable phagemid yields, with only 1-10% background K07 phage, are obtained when the K07 multiplicity of infection is 100.
TABLE IV Enrichment hGHbp/BSA Phage Pool moi (K07) CFU/PFU beads Fraction Kpnl LIB 1 1000 ND 14 0.44 LIB 1G1000 ND 30 0.57 LIB 3100 ND 1.7 0.26 LIB 3G10 ND 8.5 0.18 LIB 3G100 460 220 0.13 LIB 5100 ND 15 ND LIB 2 100 ND 1.7 <0.05 LIB 2G10 ND 4.1 <0.10 LIB 2G100 1000 27 0.18 LIB 4100 170 38 ND - Phage pools are labelled as shown (
FIG. 3 ). The multiplicity of infection (moi) refers to the multiplicity of K07 infection (PFU/cells) in the propagation of phagemid. The enrichment of CFU over PFU is shown in those cases where purified K07 was added in the binding step. The ratio of CFU eluting from hGHbp-beads over CFU eluting from BSA-beads is shown. The fraction of KpnI-containing template (i.e., pH0415) remaining in the pool was determined by digesting dsDNA with KpnI plus EcoRI, running the products on a 1% agarose gel, and laser-scanning a negative of the ethidium bromide-stained DNA. - Receptor-Binding Affinity of the Hormone hGH(E174S, F176Y)
- The fact that a single clone was isolated from two different pathways of selection (
FIG. 3 ) suggested that the double mutant (E174S,F176Y) binds strongly to hGHbp. To determine the affinity of this mutant of hGH for hGHbp, we constructed this mutant of hGH by site-directed mutagenesis, using a plasmid (pB0720) which contains the wild-type hGH gene as template and the following oligonucleotide which changescodons 174 and 176 (SEQ ID NO:10):hGH codon: 172 174 176 178 Lys Ser Tyr Arg 5′-ATG GAC AAG GTG TCG ACA TAC CTG CGC ATC GTG-3′
The resulting construct, pH0458B, was transformed into E. coli strain 16C9 for expression of the mutant hormone. Scatchard analysis of competitive binding of hGH(E174S,F176Y) versus 125I-hGH to hGHbp indicated that the (E174S,F176Y) mutant has a binding affinity at least 5.0-fold tighter than that of wild-type hGH. - Human growth hormone variants were produced by the method of the present invention using the phagemid described in
FIG. 9 . - Construction of a De-Fusable Hormone-Phage Vector
- We designed a vector for cassette mutagenesis (Wells et al., Gene 34, 315-323 [1985]) and expression of the hGH-gene III fusion protein with the objectives of (1) improving the linkage between hGH and the gene III moiety to more favorably display the hGH moiety on the phage (2) limiting expression of the fusion protein to obtain essentially “monovalent display,” (3) allowing for restriction nuclease selection against the starting vector, (4) eliminating expression of fusion protein from the starting vector, and (5) achieving facile expression of the corresponding free hormone from a given hGH-gene III fusion mutant.
- Plasmid pS0643 was constructed by oligonucleotide-directed mutagenesis (Kunkel et al., Methods Enzymol. 154, 367-382 [1987]) of pS0132, which contains pBR322 and f1 origins of replication and expresses an hGH-gene III fusion protein (hGH residues 1-191, followed by a single Gly residue, fused to Pro-198 of gene III) under the control of the E. coli phoA promoter (Bass et al.,
Proteins 8, 309-314 [1990]) (FIG. 9 ). Mutagenesis was carried out with theoligonucleotide 5′-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3′ (SEQ ID NO:1), which introduces a XbaI site (underlined) and an amber stop codon (TAG) following Phe-191 of hGH. In the resulting construct, pS0643, a portion of gene III was deleted, and two silent mutations (underlined) occurred, yielding the following junction between hGH and gene III (SEQ ID NOS: 11 AND 12):--- hGH---------------> gene III---------------> 187 188 189 190 191 am* 249 250 251 252 253 254 Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly GGC AGC TGT GGA TTC TAG AGT GGC GGT GGC TCT GGT - This shortens the total size of the fusion protein from 401 residues in pS0132 to 350 residues in pS0643. Experiments using monoclonal antibodies against hGH have demonstrated that the hGH portion of the new fusion protein, assembled on a phage particle, is more accessible than was the previous, longer fusion.
- For propagation of hormone-displaying phage, pS0643 and derivatives can be grown in a amber-suppressor strain of E. coli, such as JM101 or XL1-Blue (Bullock et al.,
BioTechniques 5, 376-379 [1987]). Shown above is substitution of Glu at the amber codon which occurs in supE suppressor strains. Suppression with other amino acids is also possible in various available strains of E. coli well known and publically available. - To express hGH (or mutants) free of the gene III portion of the fusion, pS0643 and derivatives can simply be grown in a non-suppressor strain such as 16C9. In this case, the amber codon (TAG) leads to termination of translation, which yields free hormone, without the need for an independent DNA construction.
- To create sites for cassette mutagenesis, pS0643 was mutated with the oligonucleotides (1) 5′-CGG-ACT-GGG-CAG-ATA-TTC-AAG-CAG-ACC-3′ (SEQ ID NO:13), which destroys the unique BglII site of pS0643; (2) 5′-CTC-AAG-AAC-TAC-GGG-TTA-CCC-TGA-CTG-CTT-CAG-GAA-GG-3′ (SEQ ID NO:14), which inserts a unique BstEII site, a single-base frameshift, and a non-amber stop codon (TGA); and (3) 5′-CGC-ATC-GTG-CAG-TGC-AGA-TCT-GTG-GAG-GGC-3′ (SEQ ID NO:15), which introduces a new BgIII site, to yield the starting vector, pH0509. The addition of a frameshift along with a TGA stop codon insures that no gene III-fusion can be produced from the starting vector. The BstEII-BglII segment is cut out of pH0509 and replaced with a DNA cassette, mutated at the codons of interest. Other restriction sites for cassette mutagenesis at other locations in hGH have also been introduced into the hormone-phage vector.
- Cassette Mutagenesis within
Helix 4 of hGH -
Codons helix 4; and they are each substituted by at least one amino acid among known evolutionary variants of hGH. - We chose to substitute NNS(N=A/G/C/T; S=G/C) at each of the target residues. The choice of the NNS degenerate sequence yields 32 possible codons (including at least one codon for each amino acid) at 4 sites, for a total of (32)4=1,048,576 possible nucleotide sequences, or (20)4=160,000 possible polypeptide sequences. Only one stop codon, amber (TAG), is allowed by this choice of codons, and this codon is suppressible as Glu in supE strains of E. coli.
- Two degenerate oligonucleotides, with NNS at
codons - The vector was prepared by digesting pH0509 with BstEII followed by BglII. The products were run on a 1% agarose gel and the large fragment excised, phenol-extracted, and ethanol precipitated. This fragment was treated with calf intestinal phosphatase (Boehringer), then phenol:chloroform extracted, ethanol precipitated, and resuspended for ligation with the mutagenic cassette.
- Propagation of the Initial Library in XL1-Blue Cells
- Following ligation, the reaction products were again digested with BstEII, then phenol:chloroform extracted, ethanol precipitated and resuspended in water. (A BstEII recognition site (GGTNACC) is created within cassettes which contain a G at
position 3 ofcodon 172 and an ACC (Thr) codon at 174. However, treatment with BstEII at this step should not select against any of the possible mutagenic cassettes, because virtually all cassettes will be heteroduplexes, which cannot be cleaved by the enzyme.) Approximately 150 ng (45 fmols) of DNA was electroporated into XL1-Blue cells (1.8×109 cells in 0.045 mL) in a 0.2 cm cuvette at a voltage setting of 2.49 kV with a single pulse (time constant=4.7 msec.). - The cells were allowed to recover 1 hour at 37° C. in S.O.C media with shaking, then mixed with 25 mL 2YT medium, 100 mg/mL carbenicillin, and M13-K07 (moi=100). After 10′ at 23° C., the culture was incubated overnight (15 hours) at 37° C. with shaking. Plating of serial dilutions from this culture onto carbenicillin-containing media indicated that 3.9×107 electrotransformants were obtained.
- After overnight incubation, the cells were pelleted, and double-stranded DNA (dsDNA), designated pH0529E (the initial library), was prepared by the alkaline lysis method. The supernatant was spun again to remove any remaining cells, and the phage, designated phage pool φH0529E (the initial library of phage), were PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were measured as colony-forming units (CFU) for the recombinant phagemid containing hGH-g3p. Approximately 4.5×1013 CFU were obtained from the starting library.
- Degeneracy of the Starting Library
- From the pool of electrotransformants, 58 clones were sequenced in the region of the BstEII-BglII cassette. Of these, 17% corresponded to the starting vector, 17% contained at least one frame shift, and 7% contained a non-silent (non-terminating) mutation outside the four target codons. We conclude that 41% of the clones were defective by one of the above measures, leaving a total functional pool of 2.0×107 initial transformants. This number still exceeds the possible number of DNA sequences by nearly 20-fold. Therefore, we are confident of having all possible sequences represented in the starting library.
- We examined the sequences of non-selected phage to evaluate the degree of codon bias in the mutagenesis (Table V). The results indicated that, although some codons (and amino acids) are under- or over-represented relative to the random expectation, the library is extremely diverse, with no evidence of large-scale “sibling” degeneracy (Table VI).
TABLE V Codon distribution (per 188 codons) of non-selected hormone phage. Clones were sequenced from the starting library (pH0529E). All codons were tabulated, including those from clones which contained spurious mutations and/or frameshifts. Residue Number expected Number found Found/Expected Leu 17.6 18 1.0 Ser 17.6 26 1.5 Arg 17.6 10 0.57 Pro 11.8 16 1.4 Thr 11.8 14 1.2 Ala 11.8 13 1.1 Gly 11.8 16 1.4 Val 11.8 4 0.3 Ile 5.9 2 0.3 Met 5.9 1 0.2 Tyr 5.9 1 0.2 His 5.9 2 0.3 Trp 5.9 2 0.3 Phe 5.9 5 0.9 Cys 5.9 5 0.9 Gln 5.9 7 1.2 Asn 5.9 14 2.4 Lys 5.9 11 1.9 Asp 5.9 9 1.5 Glu 5.9 6 1.0 amber* 5.9 6 1.0
*Note: the amber stop codon (TAG) is suppressed as Glu in XL1-Blue cells. Highlighted codons were over/under-represented by 50% or more.
-
TABLE VI Non-selected (pH0529E) clones with an open reading frame. The notation, e.g. TWGS, denotes the hGH mutant 172T/174W/176G/178S. Amber (TAG) codons, translated as Glu in XL1-Blue cells are shown as ε. Kε NT (SEQ ID NO:46) TWGS (SEQ ID NO:47) Pε ER (SEQ ID NO:48) LPPS (SEQ ID NO:49) SLDP (SEQ ID NO:50) QQSN (SEQ ID NO:51) GSKT (SEQ ID NO:52) TPVT (SEQ ID NO:53) RSRA (SEQ ID NO:54) LCGL (SEQ ID NO:55) TGRL (SEQ ID NO:56) AKAS (SEQ ID NO:57) GNDD (SEQ ID NO:58) KTEQ (SEQ ID NO:59) NNCR (SEQ ID NO:60) FPCL (SEQ ID NO:61) NSDF (SEQ ID NO:62) HRPS (SEQ ID NO:63) LSLε (SEQ ID NO:64) NGSK (SEQ ID NO:65) LTTE (SEQ ID NO:66) PSGG (SEQ ID NO:67) LWFP (SEQ ID NO:68) PAGS (SEQ ID NO:69) GRAK (SEQ ID NO:70) GTNG (SEQ ID NO:71) CVLQ (SEQ ID NO:72) EASL (SEQ ID NO:73) SSKE (SEQ ID NO:74) ALLL (SEQ ID NO:75) PSHP (SEQ ID NO:76) SYAP (SEQ ID NO:77) ASNG (SEQ ID NO:78) EANN (SEQ ID NO:79) KNAK (SEQ ID NO:80) SRGK (SEQ ID NO:81) GLDG (SEQ ID NO:82) NDPI (SEQ ID NO:83)
Preparation of Immobilized hGHbp and hPRLbp - Immobilized hGHbp (“hGHbp-beads”) was prepared as described (Bass et al.,
Proteins 8, 309-314 [1990]), except that wild-type hGHbp (Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]) was used. Competitive binding experiments with [125I] hGH indicated that 58 fmols of functional hGHbp were coupled per μL of bead suspension. - Immobilized hPRLbp (“hPRLbp-beads”) was prepared as above, using the 211-residue extracellular domain of the prolactin receptor (Cunningham et al.,
Science 250, 1709-1712 [1990]). Competitive binding experiments with [125I] hGH in the presence of 50 μM zinc indicated that 2.1 fmols of functional hPRLbp were coupled per μL of bead suspension. - “Blank beads” were prepared by treating the oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) for 15 hours at 4° C.
- Binding Selection Using Immobilized hGHbp and hPRLbp
- Binding of hormone-phage to beads was carried out in one of the following buffers: Buffer A (PBS, 0.5% BSA, 0.05
% Tween 20, 0.01% thimerosal) for selections using hGHbp and blank beads; Buffer B (50 mM tris pH 7.5, 10 mM MgCl2, 0.5% BSA, 0.05% Tween % Tween 20, 0.01% thimerosal, 10 mM EDTA) for selections using hPRLbp in the absence of zinc (+EDTA). Binding selections were carried out according to each of the following paths: (1) binding to blank beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads (+Zn2+), (4) binding to hPRLbp-beads (+EDTA), (5) pre-adsorbing twice with hGHbp beads then binding the non-adsorbed fraction to hPRLbp-beads (“−hGHbp, +hPRLbp” selection), or (6) pre-adsorbing twice with hPRLbp-beads then binding the non-adsorbed fraction to hGHbp-beads (“−hPRLbp, +hGHbp” selection). The latter two procedures are expected to enrich for mutants binding hPRLbp but not hGHbp, or for mutants binding hGHbp but not hPRLbp, respectively. Binding and elution of phage was carried out in each cycle as follows: - 1. BINDING: An aliquot of hormone phage (typically 109-1010 CFU) was mixed with an equal amount of non-hormone phage (pCAT), diluted into the appropriate buffer (A, B, or C), and mixed with a 10 mL suspension of hGHbp, hPRLbp or blank beads in a total volume of 200 mL in a 1.5 mL polypropylene tube. The phage were allowed to bind to the beads by incubating 1 hour at room temperature (23° C.) with slow rotation (approximately 7 RPM). Subsequent steps were carried out with a constant volume of 200 μL and at room temperature.
- 2. WASHES: The beads were spun 15 sec., and the supernatant was removed. To reduce the number of phage not specifically bound, the beads were washed 5 times by resuspending briefly in the appropriate buffer, then pelleting.
- 3. hGH ELUTION: Phage binding weakly to the beads were removed by elution with hGH. The beads were rotated with the appropriate buffer containing 400 nM hGH for 15-17 hours. The supernatant was saved as the “hGH elution” and the beads. The beads were washed by resuspending briefly in buffer and pelleting.
- 4. GLYCINE ELUTION: To remove the tightest-binding phage (i.e. those still bound after the hGH wash), beads were suspended in Glycine buffer (Buffer A plus 0.2 M Glycine, pH 2.0 with HCl), rotated 1 hour and pelleted. The supernatant (“Glycine elution”; 200 μL) was neutralized by adding 30 mL of 1 M Tris base and stored at 4° C.
- 5. PROPAGATION: Aliquots from the hGH elutions and from the Glycine elutions from each set of beads under each set of conditions were used to infect separate cultures of log-phase XL1-Blue cells. Transductions were carried out by mixing phage with 1 mL XL1-Blue cells, incubating 20 min. at 37° C., then adding K07 (moi=100). Cultures (25 mL 2YT plus carbenicillin) were grown as described above and the next pool of phage was prepared as described above.
- Phage binding, elution, and propagation were carried out in successive rounds, according to the cycle described above. For example, the phage amplified from the hGH elution from hGHbp-beads were again selected on hGHbp-beads and eluted with hGH, then used to infect a new culture of XL1-Blue cells. Three to five rounds of selection and propagation were carried out for each of the selection procedures described above.
- DNA Sequencing of Selected Phagemids
- From the hGH and Glycine elution steps of each cycle, an aliquot of phage was used to inoculate XL1-Blue cells, which were plated on LB media containing carbenicillin and tetracycline to obtain independent clones from each phage pool. Single-stranded DNA was prepared from isolated colony and sequenced in the region of the mutagenic cassette. The results of DNA sequencing are summarized in terms of the deduced amino acid sequences in
FIGS. 5, 6 , 7, and 8. - Expression and Assay of hGH Mutants
- To determine the binding affinity of some of the selected hGH mutants for the hGHbp, we transformed DNA from sequenced clones into E. coli strain 16C9. As described above, this is a non-suppressor strain which terminates translation of protein after the final Phe-191 residue of hGH. Single-stranded DNA was used for these transformations, but double-stranded DNA or even whole phage can be easily electroporated into a non-suppressor strain for expression of free hormone.
- Mutants of hGH were prepared from osmotically shocked cells by ammonium sulfate precipitation as described for hGH (Olson et al., Nature 293, 408-411 [1981]), and protein concentrations were measured by laser densitomoetry of Coomassie-stained SDS-polyacrylamide gel electrophoresis gels, using hGH as standard (Cunningham and Wells, Science 244, 1081-1085 [1989]).
- The binding affinity of each mutant was determined by displacement of 125I hGH as described (Spencer et al., J. Biol. Chem. 263, 7862-7867 [1988]; Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]), using an anti-receptor monoclonal antibody (Mab263).
- The results for a number of hGH mutants, selected by different pathways (
FIG. 6 ) are shown in Table VII. Many of these mutants have a tighter binding affinity for hGHbp than wild-type hGH. The most improved mutant, KSYR (SEQ ID NO:84), has a binding affinity 5.6 times greater than that of wild-type hGH. The weakest selected mutant, among those assayed was only about 10-fold lower in binding affinity than hGH. - Binding assays may be carried out for mutants selected for hPRLbp-binding.
TABLE VII Competitive binding to hGHbp The selected pool in which each mutant was found is indicated as 1G (first glycine selection), 3G (third glycine selection), 3H (third hGH selection), 3* (third selection, not binding to hPRLbp, but binding to hGHbp). The number of times each mutant occurred among all sequenced clones is shown ( ). Kd(mut)/ Mutant Kd (nM) Kd(hGH) Pool KSYR (6) (SEQ ID NO: 84) 0.06 + 0.01 0.18 1G, 3G RSFR (SEQ ID NO: 85) 0.10 + 0.05 0.30 3G RAYR (SEQ ID NO: 86) 0.13 + 0.04 0.37 3* KTYK (2) (SEQ ID NO: 87) 0.16 + 0.04 0.47 H, 3G RSYR (3) (SEQ ID NO: 88) 0.20 + 0.07 0.58 1G, 3H, 3G KAYR (3) (SEQ ID NO: 89) 0.22 + 0.03 0.66 3G RFFR (2) (SEQ ID NO: 90) 0.26 + 0.05 0.76 3H KQYR (SEQ ID NO: 91) 0.33 + 0.03 1.0 3G KEFR = wt (9) 0.34 + 0.05 1.0 3H, 3G, 3* RTYH (SEQ ID NO: 92) 0.68 + 0.17 2.0 3H QRYR (SEQ ID NO: 93) 0.83 + 0.14 2.5 3* KKYK (SEQ ID NO: 94) 1.1 + 0.4 3.2 3* RSFS (2)(SEQ ID NO: 95) 1.1 + 0.2 3.3 3G, * KSNR (SEQ ID NO: 96) 3.1 + 0.4 9.2 3*
Additive and Non-Additive Effects on Binding - At some residues, substitution of a particular amino acid has essentially the same effect independent of surrounding residues. For example, substitution of F176Y in the background of 172R/174S reduces binding affinity by 2.0-fold (RSFR (SEQ ID NO:85) vs. RSYR (SEQ ID NO:88)). Similarly, in the background of 172K/174A the binding affinity of the F176Y mutant (KAYR (SEQ ID NO:89)) is 2.9-fold weaker than the corresponding 176F mutant (KAFR; Cunningham and Wells, 1989).
- On the other hand, the binding constants determined for several selected mutants of hGH demonstrate non-additive effects of some amino acid substitutions at
residues - Such non-additive effects on binding for substitutions at proximal residues illustrate the utility of protein-phage binding selection as a means of selecting optimized mutants from a library randomized at several positions. In the absence of detailed structural information, without such a selection process, many combinations of substitutions might be tried before finding the optimum mutant.
- Using the methods described in Example VIII, we targeted another region of hGH involved in binding to the hGHbp and/or hPRLbp,
helix 1residues - We chose to use the “amber” hGH-g3 construct (called phGHam-g3p) because it appears to make the target protein, hGH, more accessible for binding. This is supported by data from comparative ELISA assays of monoclonal antibody binding. Phage produced from both pS0132 (S. Bass, R. Greene, J. A. Wells,
Proteins 8, 309 (1990).) and phGHam-g3 were tested with three antibodies (Medix 2, 1B5.G2, and 5B7.C10) that are known to have biriding determinants near the carboxyl-terminus of hGH [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); L. Jin and J. Wells, unpublished results], and one antibody (Medix 1) that recognizes determinants inhelices 1 and 3 ([B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)]). Phagemid particles from phGHam-g3 reacted much more strongly withantibodies Medix 2, 1B5.G2, and 5B7.C10 than did phagemid particles from pS0132. In particular, binding of pS0132 particles was reduced by >2000-fold for bothMedix 2 and 5B7.C10 and reduced by >25-fold for 1B5.G2 compared to binding toMedix 1. On the other hand, binding of phGHam-g3 phage was weaker by only about 1.5-fold, 1.2-fold, and 2.3-fold for theMedix 2, 1B5.G2, and 5B7.C10 antibodies, respectively, compared with binding toMEDIX 1. - Construction of the
Helix 1 Library by Cassette Mutagenesis - We mutated residues in
helix 1 that were previously identified by alanine-scanning mutagenesis [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989), 15, 16) to modulate the binding of the extracellular domains of the hGH and/or hPRL receptors (called hGHbp and hPRLbp, respectively). Cassette mutagenesis was carried out essentially as described [J. A. Wells, M. Vasser, D. B. Powers, Gene 34, 315 (1985)]. This library was constructed by cassette mutagenesis that fully mutated four residues at a time (see Example VIII) which utilized a mutated version of phGHam-g3 into which unique KpnI (at hGH codon 27) and XhoI (at hGH codon 6) restriction sites (underlined below) had been inserted by mutagenesis [T. A. Kunkel, J. D. Roberts, R. A. Zakour, Methods Enzymol. 154, 367-382] with theoligonucleotides 5′-GCC TTT GAC AGG TAC CAG GAG m G-3′ (SEQ ID NO:18) and 5′-CCA ACT ATA CCA CTC TCG AGG TCT ATT CGA TAA C-3′ (SEQ ID NO:19), respectively. The later oligo also introduced a+1 frameshift (italicized) to terminate translation from the starting vector and minimize wild-type background in the phagemid library. This strating vector was designated pH0508B. Thehelix 1 library, whichmutated hGH residues complementary oligonucleotides 5′-pTCG AGG CTC NNS GAC AAC GCG NNS CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3′ (SEQ ID NO:20) and 5′-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG SNN CGC GTT GTC SNN GAG CC-3′ (SEQ ID NO:21). The KpnI site was destroyed in the junction of the ligation product so that restriction enzyme digestion could be used for analysis of non-mutated background. - The library contained at least 107 independent transformants so that if the library were absolutely random (106 different combinations of codons) we would have an average of about 10 copies of each possible mutated hGH gene. Restriction analysis using KpnI indicated that at least 80% of
helix 1 library constructs contained the inserted cassette. - Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). Four residues in helix 1 (F10, M14, H18, and H21) were similarly mutated and after 4 and 6 cycles a non-wild-type consensus developed (Table VIII).
Position 10 on the hydrophobic face ofhelix 1 tended to be hydrophobic whereaspositions 21 and 18 on the hydrophillic face tended were dominated by Asn; no obvious consensus was evident for position 14 (Table IX). - The binding constants for these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, several mutants bind tighter to hGHbp than does wt-hGH.
TABLE VIII Selection of hGH helix 1 mutantsVariants of hGH (randomly mutated at residues F10, M14, H18, H21) expressed on phagemid particles were selected by binding to hGHbp-beads and eluting with hGH (0.4 mM) buffer followed by glycine (0.2 M, pH 2) buffer (see Example VIII). Gly elution F10 M14 H18 H21 4 Cycles H G N N A W D N (2) Y T V N I N I N L N S H F S F G 6 Cycles H G N N (6) F S F L Consensus: H G N N -
TABLE IX Consensus sequences from the selected helix 1 libraryObserved frequency is fraction of all clones sequenced with the indicated amino acid. The nominal frequency is calculated on the basis of NNS 32 codon degeneracy. The maximal enrichment factor varies from 11 to 32 depending upon the nominal frequency value for a given residue. Values of [Kd(Ala mut)/Kd(wt hGH)] for single alanine mutations were taken from B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991). Frequency Wild type residue Selected residue observed nominal Enrichment F10 5.9 H 0.50 0.031 17 F 0.14 0.031 5 A 0.14 0.062 2 M14 2.2 G 0.50 0.062 8 W 0.14 0.031 5 N 0.14 0.031 5 S 0.14 0.093 2 H18 1.6 N 0.50 0.031 17 D 0.14 0.031 5 F 0.14 0.031 5 H21 0.33 N 0.79 0.031 26 H 0.07 0.031 2 -
TABLE X Binding of purified hGH helix 1 mutants to hGHbpCompetition binding experiments were performed using [125I]hGH (wild-type), hGHbp (containing the extracellular receptor domain, residues 1-238), and Mab263 [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989)];. The number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection. Sequence position 10 14 18 21 P Kd (nM)\f (Kd mut) Kd (wt hGH)) H G N N 0.50 0.14 ± 0.04 0.42 A W D N 0.14 0.10 ± 0.03 0.30 wt = F M H H 0 0.34 ± 0.05 (1) F S F L 0.07 0.68 ± 0.19 2.0 Y T V N 0.07 0.75 ± 0.19 2.2 L N S H 0.07 0.82 ± 0.20 2.4 I N I N 0.07 1.2 ± 0.31 3.4 - Design of mutant proteins with improved binding properties by iterative selection using hormone-phage
- Our experience with recruiting non-binding homologs of hGH evolutionary variants suggests that many individual amino acid substitutions can be combined to yield cumulatively improved mutants of hGH with respect to binding a particular receptor [B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991); H. B. Lowman, B. C. Cunningham, J. A. Wells, J. Biol. Chem. 266, in press (1991)].
- The helix 4b library was constructed in an attempt to further improve the
helix 4 double mutant (E174S/F176Y) selected from the helix 4a library that we found bound tighter to the hGH receptor (see Example VIII). With the E174S/F176Y hGH mutant as the background starting hormone, residues were mutated that surroundedpositions - Construction of the Helix 4b Library by Cassette Mutagenesis
- Cassette mutagenesis was carried out essentially as described [J. A. Wells, M. Vasser, D. B. Powers, Gene 34, 315 (1985)]. The helix 4b library, which
mutated residues complementary oligonucleotides 5′-pG TTA CTC TAC TGC TTC NNS MG GAC ATG NNS MG GTC AGC NNS TAC CTG CGC NNS GTG CAG TGC A-3′ (SEQ ID NO:22) and 5′-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC CTT SNN CAT GTC CTT SNN GM GCA GTA GA-3′ (SEQ ID NO:23). The BstEII site was eliminated in the ligated cassette. From the helix 4b library, 15 unselected clones were sequenced. Of these, none lacked a cassette insert, 20% were frame-shifted, and 7% had a non-silent mutation. - Results of hGHbp Enrichment
- Binding enrichments of hGH-phage from the libraries was carried out using hGHbp immobilized on oxirane-polyacrylamide beads (Sigma Chemical Co.) as described (Example VIII). After 6 cycles of binding a reasonably clear consensus developed (Table XI). Interestingly, all positions tended to contain polar residues, notably Ser, Thr and Asn (XII).
- Assay of hGH Mutants
- The binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII). As indicated, the binding affinities of several helix-4-b mutants for hGHbp were tighter than that of wt-hGH Table XIII).
- Receptor-Selectivity of hGH Variants
- Finally, we have begun to analyze the binding affinity of several of the tighter hGHbp binding mutants for their ability to bind to the hPRLbp. The E174S/F176Y mutant binds 200-fold weaker to the hPRLbp than hGH. The E174T/F176Y/R178K and R167N/D171 S/E174S/F176Y/1179T mutants each bind >500-fold weaker to the hPRLbp than hGH. Thus, it is possible to use the produce new receptor selective mutants of hGH by phage display technology.
- Hormone-Phagemid Selection Identifies the Information-Content of Particular Residues
- Of the 12 residues mutated in three hGH-phagemid libraries (Examples VIII, IX, X), 4 showed a strong, although not exclusive, conservation of the wild-type residues (K172, T175, F176, and R178). Not surprisingly, these were residues that when converted to Ala caused the largest disruptions (4- to 60-fold) in binding affinity to the hGHbp. There was a class of 4 other residues (F10, M14, D171, and I179) where Ala substitutions caused weaker effects on binding (2- to 7-fold) and these positions exhibited little wild-type consensus. Finally the other 4 residues (H18, H21, R167, and E174), that promote binding to he hPRLbp but not the hGHbp, did not exhibit any consensus for the wild-type hGH sequence by selection on hGHbp-beads. In fact two residues (E174 and H21), where Ala substitutions enhance binding affinity to the hGHbp by 2- to 4-fold [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991)]. Thus, the alanine-scanning mutagenesis data correlates reasonably well with the flexibility to substitute each position. In fact, the reduction in binding affinity caused by alanine substitutions [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)], B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991)] is a reasonable predictor of the percentage that the wild-type residue is found in the phagemid pool after 3-6 rounds of selection. The alanine-scanning information is useful for targeting side-chains that modulate binding, and the phage selection is appropriate for optimizing them and defining the flexibility of each site (and/or combinations of sites) for substitution. The combination of scanning mutational methods [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989)] and phage display is a powerful approach to designing receptor-ligand interfaces and studying molecular evolution in vitro.
- Variations on Iterative Enrichment of Hormone-Phagemid Libraries
- In cases where combined mutations in hGH have additive effects on binding affinity to receptor, mutations learned through hormone-phagemid enrichment to improve binding can be combined by simple cutting and ligation of restriction fragments or mutagenesis to yield cumulatively optimized mutants of hGH.
- On the other hand, mutations in one region of hGH which optimize receptor binding may be structurally or functionally incompatible with mutations in an overlapping or another region of the molecule. In these cases, hormone phagemid enrichment can be carried out by one of several variations on the iterative enrichment approach: (1) random DNA libraries can be generated in each of two (or perhaps more) regions of the molecule by cassette or another mutagenesis method. Thereafter, a combined library can be created by ligation of restriction fragments from the two DNA libraries; (2) an hGH variant, optimized for binding by mutation in one region of the molecule, can be randomly mutated in a second region of the molecule as in the helix-4-b library example; (3) two or more random libraries can be partially selected for improved binding by hormone-phagemid enrichment; after this “roughing-in” of the optimized binding site, the still-partially-diverse libraries can be recombined by ligation of restriction fragments to generate a single library, partially diverse in two or more regions of the molecules, which in turn can be further selected for optimized binding using hormone-phagemid enrichment.
TABLE XI Mutant phagemids of hGH selected from helix 4b library after 4 and 6 cycles of enrichment. Selection of hGH helix 4b mutants (randomly mutated at residues containing the E174S/F176Y double mutant, by binding to hGHbp-beads and eluting with hGH (0.4 mM) buffer followed by glycine (0.2 M, pH 2) buffer. One mutant (+) contained the spurious mutation R178H. R167 D171 T175 I179 4 Cycles N S T T K S T T S N T T D S T T D S T T+ D S A T D S A N T D T T N D T N A N T N A S T T 6 Cycles N S T T (2) N N T T N S T Q D S S T E S T I K S T L Consensus: N S T T D N -
TABLE XII Consensus sequences from the selected library. Observed frequency is fraction of all clones sequenced with the indicated amino acid. The nominal frequency is calculated on the basis of NNS 32 codon degeneracy. The maximal enrichment factor varies from 11 to 16 to 32 depending upon the nominal frequency value for a given residue. Values of [Kd(Ala mut)/Kd(wt hGH)] for single alanine mutations were taken from refs. below; for position 175 we onlyhave a value for the T175S mutant [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991).]. Frequency Wild type residue Selected residue observed nominal Enrichment R167 0.75 N 0.35 0.031 11 D 0.24 0.031 8 K 0.12 0.031 4 A 0.12 0.062 2 D171 7.1 S 0.76 0.093 8 N 0.18 0.031 6 D 0.12 0.031 4 T175 3.5 T 0.88 0.062 14 A 0.12 0.031 4 I179 2.7 T 0.71 0.062 11 N 0.18 0.031 6 -
TABLE XIII Binding of purified hGH mutants to hGHbp. Competition binding experiments were performed using [125I]hGH (wild-type), hGHbp (containing the extracellular receptor domain, residues 1-238), and Mab263 (11). The number P indicates the fractional occurrence of each mutant among all the clones sequenced after one or more rounds of selection. Note that the helix 4b mutations (*) are in the background of hGH(E174S/F176Y). In the list of helix 4b mutants,, the E174S/F176Y mutant (*), with wt residues at 167, 171, 175, 179, is shown in bold. Sequence position P Kd (nM) * * * * 167 171 175 179 N S T T 0.18 0.04 ± 0.02 0.12 E S T I 0.06 0.04 ± 0.02 0.12 K S T L 0.06 0.05 ± 0.03 0.16 N N T T 0.06 0.06 ± 0.03 0.17 R D T I 0 0.06 ± 0.01 (0.18) N S T Q 0.06 0.26 ± 0.11 0.77 - Construction of Plasmids
- Plasmid pDH 188 contains the DNA encoding the Fab portion of a humanized IgG antibody, called 4D5, that recognizes the HER-2 receptor. This plasmid is contained in E. coli strain SR 101, and has been deposited with the ATCC in Rockville, Md.
- Briefly, the plasmid was prepared as follows: the starting plasmid was pS0132, containing the alkaline phosphatase promoter as described above. The DNA encoding human growth hormone was excised and, after a series of manipulations to make the ends of the plasmid compatible for ligation, the DNA encoding 4D5 was inserted. The 4D5 DNA contains two genes. The first gene encodes the variable and constant regions of the light chain, and contains at its 5′ end the DNA encoding the st II signal sequence. The second gene contains four portions: first, at its 5′ end is the DNA encoding the st II signal sequence. This is followed by the DNA encoding the variable domain of the heavy chain, which is followed by the DNA encoding the first domain of the heavy chain constant region, which in turn is followed by the DNA encoding the M13 gene III. The salient features of this construct are shown in
FIG. 10 . The sequence of the DNA encoding 4D5 is shown inFIG. 11 . - E. coli Transformation and Phage Production.
- Both polyethylene glycol (PEG) and electroporation were used to transform plasmids into SR101 cells. (PEG competent cells were prepared and transformed according to the method of Chung and Miller (Nucleic Acids Res. 16:3580 [1988]). Cells that were competent for electroporation were prepared, and subsequently transformed via electroporation according to the method of Zabarovsky and Winberg (Nucleic Acids Res. 18:5912 [1990]). After placing the cells in 1 ml of the SOC media (described in Sambrook et al., supra), they were grown for 1 hour at 37° C. with shaking. At this time, the concentration of the cells was determined using light scattering at OD600. A
titered K 7 phage stock was added to achieve an multiplicity of infection (MOI) of 100, and the phage were allowed to adhere to the cells for 20 minutes at room temperature. This mixture was then diluted into 25 mls of 2YT broth (described in Sambrook et al, supra) and incubated with shaking at 37° C. overnight. The next day, cells were pelleted by centrifugation at 5000×g for 10 minutes, the supernatant was collected, and the phage particles were precipitated with 0.5 M NaCl and 4% PEG (final concentration) at room temperature for 10 minutes. Phage particles were pelleted by centrifugation at 10,000×g for 10 minutes, resuspended in 1 ml of TEN (10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl), and stored at 4° C. - Production of Antigen Coated Plates.
- Aliquots of 0.5 ml from a solution of 0.1 mg/ml of the extra-cellular domain of the HER-2 antigen (ECD) or a solution of 0.5 mg/ml of BSA (control antigen) in 0.1 M sodium bicarbonate, pH 8.5 were used to coat one well of a
Falcon 12 well tissue culture plate. Once the solution was applied to the wells, the plates were incubated at 4° C. on a rocking platform overnight. The plates were then blocked by removing the initial solution, applying 0.5 ml of blocking buffer (30 mg/ml BSA in 0.1 M sodium bicarbonate), and incubating at room temperature for one hour. Finally, the blocking buffer was removed, 1 ml of buffer A (PBS, 0.5% BSA, and 0.05% Tween-20) was added, and the plates were stored up to 10 days at 4° C. before being used for phage selection. - Phage Selection Process.
- Approximately 109 phage particles were mixed with a 100-fold excess of
K 7 helper phage and 1 ml of buffer A. This mixture was divided into two 0.5 ml aliquots; one of which was applied to ECD coated wells, and the other was applied to BSA coated wells. The plates were incubated at room temperature while shaking for one to three hours, and were then washed three times over a period of 30 minutes with 1 ml aliquots of buffer A. Elution of the phage from the plates was done at room temperature by one of two methods: 1) an initial overnight incubation of 0.025 mg/ml purified Mu4D5 antibody (murine) followed by a 30 minute incubation with 0.4 ml of the acid elution buffer (0.2 M glycine, pH 2.1, 0.5% BSA, and 0.05% Tween-20), or 2) an incubation with the acid elution buffer alone. Eluates were then neutralized with 1 M Tris base, and a 0.5 ml aliquot of TEN was added. These samples were then propagated, titered, and stored at 4° C. - Phage Propagation
- Aliquots of eluted phage were added to 0.4 ml of 2YT broth and mixed with approximately 108 mid-log phase male E. coli strain SR101. Phage were allowed to adhere to the cells for 20 minutes at room temperature and then added to 5 ml of 2YT broth that contained 50 μg/ml of carbenicillin and 5 μg/ml of tetracycline. These cells were grown at 37° C. for 4 to 8 hours until they reached mid-log phase. The OD600 was determined, and the cells were superinfected with
K 7 helper phage for phage production. Once phage particles were obtained, they were titered in order to determine the number of colony forming units (cfu). This was done by taking aliquots of serial dilutions of a given phage stock, allowing them to infect mid-log phase SR101, and plating on LB plates containing 50 υg/ml carbenicillin. - RIA Affinity Determination.
- The affinity of h4D5 Fab fragments and Fab phage for the ECD antigen was determined using a competitive receptor binding RIA (Burt, D. R., Receptor Binding in Drug Research. O'Brien, R. A. (Ed.). pp. 3-29, Dekker, New York [1986]). The ECD antigen was labeled with 125-Iodine using the sequential chloramine-T method (De Larco, J. E. et al., J. Cell. Physiol. 109:143-152 [1981]) which produced a radioactive tracer with a specific activity of 14 μCi/μg and incorporation of 0.47 moles of Iodine per mole of receptor. A series of 0.2 ml solutions containing 0.5 ng (by ELISA) of Fab or Fab phage, 50 nCi of 125I ECD tracer, and a range of unlabeled ECD amounts (6.4 ng to 3277 ng) were prepared and incubated at room temperature overnight. The labeled ECD-Fab or ECD-Fab phage complex was separated from the unbound labeled antigen by forming an aggregate complex induced by the addition of an anti-human IgG (Fitzgerald 40-GH23) and 6% PEG 8000. The complex was pelleted by centrifugation (15,000×g for 20 minutes) and the amount of labeled ECD (in cpm) was determined by a gamma counter. The dissociation constant (Kd) was calculated by employing a modified version of the program LIGAND (Munson, P. and Rothbard, D., Anal. Biochem. 107:220-239 [1980]) which utilizes Scatchard analysis (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672 [1949]). The Kd values are shown in
FIG. 13 . - Competitive Cell Binding Assay
- Murine 4D5 antibody was labeled with 125-I to a specific activity of 40-50 μCi/μg using the lodogen procedure. Solutions containing a constant amount of labeled antibody and increasing amounts of unlabeled variant Fab were prepared and added to near confluent cultures of SK-BR-3 cells grown in 96-well microtiter dishes (final concentration of labeled antibody was 0.1 nM). After an overnight incubation at 4° C., the supernatant was removed, the cells were washed and the cell associated radioactivity was determined in a gamma counter. Kd values were determined by analyzing the data using a modified version of the program LIGAND (Munson, P. and Rothbard, D., supra)
- This deposit of plasmid pDH188 ATCC no. 68663 was 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 culture for 30 years from the date of deposit. The organisms 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 progeny of the cultures 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 progeny 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 886OG 638).
- The assignee of the present application has agreed that if the cultures on deposit should die or be lost or destroyed when cultivated under suitable conditions, they will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited cultures 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.
- The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the cultures deposited, since the deposited embodiments are intended as separate illustrations of certain aspects of the invention and any cultures that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
- While the invention has necessarily been described in conjunction with preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and alterations to the subject matter set forth herein, without departing from the spirit and scope thereof. Hence, the invention can be practiced in ways other than those specifically described herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the appended claims and equivalents thereof.
- Construction of Additive Variants of hGH
- According to additivity principles [J. A. Wells, Biochemistry 29, 8509 (1990)], mutations in different parts of a protein, if they are not mutually interacting, are expected to combine to produce additive changes in the free energy of binding to another molecule (changes are additive in terms of ΔΔGbinding, or multiplicative in terms of Kd=exp[−ΔAG/RT]). Thus a mutation producing a 2-fold increase in binding affinity, when combined with a second mutation causing a 3-fold increase, would be predicted to yield a double mutant with a 6-fold increased affinity over the starting variant.
- To test whether multiple mutations obtained from hGH-phage selections would produce cumulatively favorable effects on hGHbp (hGH-binding protein; the extracellular domain of the hGH receptor) binding, we combined mutations found in the three tightest-binding variants of hGH from the helix-1 library (Example IX: F10A/M14W/H18D/H21N, F10H/M14G/H18N/H21N, and F10F/M14S/H18F/H21L) with those found in the three tightest binding variants found in the helix-4-b library (Example X: R167N/D171S/T175/I179T, R167E/D171S/T175/I179, and R167N/D171N/T175/I179T).
- hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix variants was isolated and digested with the restriction enzymes EcoRI and BstXI. The large fragment from each helix-4-b variant was then isolated and ligated with the small fragment from each helix-1 variant to yield the new two-helix variants shown in Table XIII. All of these variants also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details).
- Construction of Selective Combinatorial Libraries of hGH
- Although additivity principles appear to hold for a number of combinations of mutations, some combinations (e.g. E174S with F176Y) are clearly non-additive (see examples VIII and X). In order to identify with certainty the tightest binding variant with, for example, 4 mutations in helix-1 and 4 mutations in helix-4, one would ideally mutate all 8 residues at once and then sort the pool for the globally tightest binding variant. However, such a pool would consist of 1.1×1012 DNA sequences (utilizing NNS codon degeneracy) encoding 2.6×1010 different polypeptides. Obtaining a random phagemid library large enough to assure representation of all variants (perhaps 1013 transformants) is not practical using current transformation technology.
- We have addressed this difficulty first by utilizing successive rounds of mutagenesis, taking the tightest binding variant from one library, then mutating other residues to further improve binding (Example X). In a second method, we have utilized the principle of additivity to combine the best mutations from two independently sorted libraries to create multiple mutants with improved binding (described above). Here, we further searched through the possible combinations of mutations at
positions helix 1 library (independently sorted for 0, 2, or 4 cycles; Example IX) and the pool from the helix-4-b library (independently sorted for 0, 2, or 4 cycles; Example X) and sorted the combined variant pool for hGHbp binding. Since some amount of sequence diversity exists in each of these pools, the resulting combinatorial library can explore more sequence combinations than what we might construct manually (e.g. Table XIII). - hGH-phagemid double-stranded DNA (dsDNA) from each of the one-helix library pools (selected for 0, 2, or 4 rounds) was isolated and digested with the restriction enzymes AccI and BstXI. The large fragment from each helix-1 variant pool was then isolated and ligated with the small fragment from each helix-4-b variant pool to yield the three combinatorial libraries pH0707A (unselected
helix 1 and helix 4b pools, as described in examples IX and X), pH0707B (twice-selected helix-1 pool with twice-selected helix-4-b pool), and pH0707C (4-times selected helix-1 pool with 4-times selected helix-4-b pool). Duplicate ligations were also set up with less DNA and designated as pH0707D, pH0707E, and pH0707F, corresponding to the 0-, 2-, and 4-round starting libraries respectively. All of these variant pools also contained the mutations E174S/F176Y obtained in earlier hGH-phage binding selections (see Example X for details). - Sorting Combinatorial Libraries of hGH-Phage Variants
- The ligation products pH0707A-F were processed and electro-transformed into XL1-Blue cells as described (Example VIII). Based on colony-forming units (CFU), the number of transformants obtained from each pool was as follows: 2.4×106 from pH0707A, 1.8×106 from pH0707B, 1.6×106 from pH0707C, 8×105 from pH0707D, 3×105 from pH0707E, and 4×105 from pH0707F. hGH-phagemid particles were prepared and selected for hGHbp-binding over 2 to 7 cycles as described in Example VIII.
- Rapid Sorting of hGH-Phagemid Libraries
- In addition to sorting phagemid libraries for tight-binding protein variants, as measured by equilibrium binding affinity, it is of interest to sort for variants which are altered in either the on-rate (kon) or the off-rate (koff) of binding to a receptor or other molecule. From thermodynamics, these rates are related to the equilibrium dissociation constant, Kd=(koff/kon). We envision that certain variants of a particular protein have similar Kd's for binding while having very different kon's and koff's. Conversely, changes in Kd from one variant to another may be due to effects on kon, effects on koff, or both. The pharmacological properties of a protein may be dependent on binding affinity or on kon or koff, depending on the detailed mechanism of action. Here, we sought to identify hGH variants with higher on-rates to investigate the effects of changes in kon. We envision that the selection could alternatively be weighted toward koff by increasing the binding time and increasing the wash time and/or concentration with cognate ligand (hGH).
- From time-course analysis of wild-type hGH-phagemid binding to immobilized hGHbp, it appears that, of the total hGH-phagemid particles that can be eluted in the
final pH 2 wash (see Example VIII for the complete binding and elution protocol), less than 10% are bound after 1 minute of incubation, while greater than 90% are bound after 15 minutes of incubation. - For “rapid-binding selection,” phagemid particles from the pH0707B pool (twice-selected for
helices - Assay of hGH Mutants
- The binding constants for some of these mutants of hGH to hGHbp was determined by expressing the free hormone variants in the non-suppressor E. coli strain 16C9 or 34B8, purifying the protein, and assaying by competitive displacement of labelled wt-hGH from hGHbp (see Example VIII) in a radio-immunoprecipitation assay. In Table XIII below, all the variants have glutamate174 replaced by serine174 and phenylalanine176 replaced by tyrosine176 (E174S and F1176Y) plus the additional substitutions as indicated at hGH amino acid positions 10, 14, 18, 21, 167, 171, 175 and 179.
TABLE XIII-A hGH variants from addition of helix-1 and helix- 4b mutations Helix 1 Helix 4wild-type residue: Variant F10 M14 H18 H21 R167 D171 T175 I179 H0650AD H G N N N S T T H0650AE H G N N E S T I H0650AF H G N N N N T T H0650BD A W D N N S T T H0650BE A W D N E S T I H0650BF A W D N N N T T H0650CD F S F L N S T T H0650CD F S F L E S T I H0650CD F S F L N N T T - In Table XIV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XIV contain two background mutations (E174S/F176Y). hGH-phagemid pools from the libraries pH0707A (Part A), pH0707B and pH0707E (Part B), or pH0707C (Part C) were sorted for 2 to 7 cycles for binding to hGHbp. The number P indicates the fractional occurrence of each variant type among the set of clones sequenced from each pool.
TABLE XIV hGH variants from hormone-phagemid binding selection of combinatorial libraries. Helix 1 Helix 4 wild-type residue: P Variant F10 M14 H18 H21 R167 D171 T175 I179 Part A: 4 cycles: 0.60 H0714A.1 H G N N N S T N 0.40 H0714A.4 A N D A N N T N* Part B: 2 cycles: 0.13 H0712B.1 F S F G H S T T 0.13 H0712B.2 H Q T S A D N S 0.13 H0712B.4 H G N N N A T T 0.13 H0712B.5 F S F L S D T T 0.13 H0712B.6 A S T N R D T I 0.13 H0712B.7 Q Y N N H S T T 0.13 H0712B.8 W G S S R D T I 0.13 H0712E.1 F L S S K N T V 0.13 H0712E.2 W N N S H S T T 0.13 H0712E.3 A N A S N S T T 0.13 H0712E.4 P S D N R D T I 0.13 H0712E.5 H G N N N N T S 0.13 H0712E.6 F S T G R D T I 0.13 H0712E.7 M T S N Q S T T 0.13 H0712E.8 F S F L T S T S 4 cycles: 0.17 H0714B.1 A W D N R D T I 0.17 H0714B.2 A W D N H S T N 0.17 H0714B.3 M Q M N N S T T 0.17 H0714B.4 H Y D H R D T T 0.17 H0714B.5 L N S H R D T I 0.17 H0714B.6 L N S H T S T T 7 cycles: 0.57 H0717B.1 A W D N N A T T 0.14 H0717B.2 F S T G R D T I 0.14 H0717B.6 A W D N R D T I 0.14 H0717B.7 I Q E H N S T T 0.50 H0717E.1 F S L A N S T V Part C: 4 cycles: 0.67 H0714C.2 F S F L K D T T
*= also contained the mutations L15R, K168R.
- In Table XV below, hGH variants were selected from combinatorial libraries by the phagemid binding selection process. All hGH variants in Table XV contain two background mutations (E174S/F176Y). The number P is the fractional occurrence of a given variant among all clones sequenced after 4 cycles of rapid-binding selection.
TABLE XV hGH variants from RAPID hGHbp binding selection of an hGH-phagemid combinatorial library Helix 1 Helix 4wild-type residue: P Variant F10 M14 H18 H21 R167 D171 T175 I179 0.14 H07BF4.2 W G S S R D T I 0.57 H07BF4.3 M A D N N S T T 0.14 H07BF4.6 A W D N S S V T‡ 0.14 H07BF4.7 H Q T S R D T I
‡= also contained the mutation Y176F (wild-type hGH also contains F176).
- In table XVI below, binding constants were measured by competitive displacement of 125I-labelled hormone H0650BD or labelled hGH using hGHbp (1-238) and either Mab5 or Mab263. The variant H0650BD appears bind more than 30-fold tighter than wild-type hGH.
TABLE XVI Equilibrium binding constants of selected hGH variants. hGH Kd(variant) Kd(variant) Variant Kd(H0650BD) Kd(hGH) Kd (pM) hGH 32 -1- 340 ± 50 H0650BD -1- 0.031 10 ± 3 H0650BF 1.5 0.045 15 ± 5 H0714B.6 3.4 0.099 34 ± 19 H0712B.7 7.4 0.22 74 ± 30 H0712E.2 16 0.48 60 ± 70 - As described in Example I, the plasmid pS0132 contains the gene for hGH fused to the residue Pro198 of the gene III protein with the insertion of an extra glycine residue. This plasmid may be used to produce hGH-phage particles in which the hGH-gene III fusion product is displayed monovalently on the phage surface (Example IV). The fusion protein comprises the entire hGH protein fused to the carboxy terminal domain of gene III via a flexible linker sequence.
- To investigate the feasibility of using phage display technology to select favourable substrate sequences for a given proteolytic enzyme, a genetically engineered variant of subtilisin BPN′ was used. (Carter, P. et al., Proteins: Structure, function and genetics 6:240-248 (1989)). This variant (hereafter referred to as A64SAL subtilisin) contains the following mutations: Ser24Cys, His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme lacks the essential catalytic residue His64, its substrate specificity is greatly restricted so that certain histidine-containing substrates are preferentially hyrdrolysed (Carter et al., Science 237:394-399 (1987)).
- Construction of a hGH-Substrate-Phage Vector
- The sequence of the linker region in pS0132 was mutated to create a substrate sequence for A64SAL subtilisin, using the
oligonucleotide 5′-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3′ (SEQ ID NO:27). This resulted in the introduction of the protein sequence Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp (SEQ ID NO:107) in the linker region between hGH and the carboxy terminal domain of gene III, where the first Phe residue in the above sequence is Phe191 of hGH. The sequence Ala-Ala-His-Tyr-Thr-Agr-Gln (SEQ ID NO:97) is known to be a good substrate for A64SAL subtilisin (Carter et al (1989), supra). The resulting plasmid was designated pS0640. - Selective Enrichment of hGH-Substrate-Phage
- Phagemid particles derived from pS0132 and pS0640 were constructed as described in Example I. In initial experiments, a 10 μl aliquot of each phage pool was separately mixed with 300 of oxirane beads (prepared as described in Example II) in 100 μl of buffer comprising 20 mM Tris-HCl pH 8.6 and 2.5M NaCl. The binding and washing steps were performed as described in example VII. The beads were then resuspended in 400 μl of the same buffer, with or without 50 nM of A64SAL subtilisin. Following incubation for 10 minutes, the supernatants were collected and the phage titres (cfu) measured. Table XVII shows that approximately 10 times more substrate-containing phagemid particles (pS0640) were eluted in the presence of enzyme than in the absence of enzyme, or than in the case of the non-substrate phagemids (pS0132) in the presence or absence of enzyme. Increasing the enzyme, phagemid or bead concentrations did not improve this ratio.
- Improvement of the Selective Enrichment Procedure
- In an attempt to decrease the non-specific elution of immobilised phagemids, a tight-binding variant of hGH was introduced in place of the wild-type hGH gene in pS0132 and pS0640. The hGH variant used was as described in example XI (pH0650bd) and contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn, Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr. This resulted in the construction of two new phagemids: pDM0390 (containing tight-binding hGH and no substrate sequence) and pDM0411 (containing tight-binding hGH and the substrate sequence Ala-Ala-His-Tyr-Thr-Agr-Gln). The binding washing and elution protocol was also changed as follows:
- (i) Binding: COSTAR 12-well tissue culture plates were coated for 16 hours with 0.5 ml/well 2 ug/ml hGHbp in sodium carbonate buffer pH 10.0. The plates were then incubated with 1 ml/well of blocking buffer (phosphate buffered saline (PBS) containing 0.1% w/v bovine serum albumen) for 2 hours and washed in an assay buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 100 mM NaCl. Phagemids were again prepared as described in Example I: the phage pool was diluted 1:4 in the above assay buffer and 0.5 ml of phage incubated per well for 2 hours.
- (ii) Washing: The plates were washed thoroughly with PBS+0.05
% Tween 20 and incubated for 30 minuted with 1 ml of this wash buffer. This washing step was repeated three times. - (iii) Eution: The plates were incubated for 10 minutes in an elution buffer consisting of 20 mM Tris-HCl pH 8.6+100 mM NaCl, then the phage were eluted with 0.5 ml of the above buffer with or without 500 nM of A64SAL subtilisin.
- Table XVII shows that there was a dramatic increase in the ratio of specifically eluted substrate-phagemid particles compared to the method previously described for pS0640 and pS0132. It is likely that this is due to the fact that the tight-binding hGH mutant has a significantly slower off-rate for binding to hGH binding protein compared to wild-type hGH.
TABLE XVII Specific elution of substrate-phagemids by A64SAL subtilisin Colony forming units (cfu) were estimated by plating out 10 μl of 10-fold dilutions of phage on 10 μl spots of XL-1 blue cells, on LB agar plates containing 50 μg/ml carbenicillinl phagemid +50 nM A64SAL no enzyme (i) Wild-type hGH gene: binding to hGHbp-oxirane beads pS0640 (substrate) 9 × 106 cfu/10 μl 1.5 × 106 cfu/10 μl pS0132 (non-substrate) 6 × 105 cfu/10 μl 3 × 105 cfu/10 μl (ii) pH0650bd mutant hGH gene: binding to hGHbp-coated plates pDM0411 (substrate) 1.7 × 105 cfu/10 μl 2 × 103 cfu/10 μl pDM0390 (non-substrate) 2 × 103 cfu/10 μl 1 × 103 cfu/10 μl - We sought to employ the selective enrichment procedure described in Example XIII to identify good substrate sequences from a library of random substrate sequences.
- Construction of a Vector for Insertion of Randomised Substrate Cassettes We designed a vector suitable for introduction of randomised substrate cassettes, and subsequent expression of a library of substrate sequences. The starting point was the vector pS0643, described in Example VIII. Site-directed mutagenesis was carried out using the
oligonucleotide 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC-GCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:28), which introduces ApaI (GGGCCC) and SalI (GTCGAC) restriction sites between hGH and Gene III. This new construct was designated pDM0253 (The actual sequence of pDM0253 is 5′-AGC-TGT-GGC-TTC-GGG-CCC-GCC-CCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3′ (SEQ ID NO:29), where the underlined base substitution is due to a spurious error in the mutagenic oligonucleotide). In addition, the tight-binding hGH variant described in example was introduced by exchanging a fragment from pDM0411 (example XIII) The resulting library vector was designated pDM0454. - Preparation of the Library Cassette Vector and Insertion of the Mutagenic Cassette
- To introduce a library cassette, pDM0454 was digested with ApaI followed by SalI, then precipitated with 13% PEG 8000+10 mM MgCl2, washed twice in 70% ethanol and resuspended This efficiently precipitates the vector but leaves the small Apa-Sal fragment in solution (Paithankar, K. R. and Prasad, K. S, N., Nucleic Acids Research 19:1346). The product was run on a 1% agarose gel and the ApaI-SalI digested vector excised, purified using a Bandprep kit (Pharmacia) and resuspended for ligation with the mutagenic cassette.
- The cassette to be inserted contained a DNA sequence similar to that in the linker region of pS0640 and pDM0411, but with the codons for the histidine and tyrosine residues in the substrate sequence replaced by randomised codons. We chose to substitute NNS (N=G/A/T/C; S=G/C) at each of the randomised positions as described in example VIII. The oligonucleotides used in the mutagenic cassettes were: 5′-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CM-3′ (coding strand) (SEQ ID NO:30) and 5′-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3′ (non-coding strand) (SEQ ID NO:31). This cassette also destroys the SalI site, so that digestion with SalI may be used to reduce the vector background. The oligonucleotides were not phosphorylated before insertion into the Apa-Sal cassette site, as it was feared that subsequent oligomerisation of a small population of the cassettes may lead to spurious results with multiple cassette inserts. Following annealing and ligation, the reaction products were phenol:chloroform extracted, ethanol precipitated and resuspended in water. Initially, no digestion with SalI to reduce the background vector was performed. Approximately 200 ng was electroporated into XL-1 blue cells and a phagemid library was prepared as described in example VIII.
- Selection of Highly Cleavable Substrates from the Substrate Library
- The selection procedure used was identical to that described for pDM0411 and pDM0390 in example XIII. After each round of selection, the eluted phage were propagated by transducing a fresh culture of XL-1 blue cells and propagating a new phagemid library as described for hGH-phage in example VIII. The progress of the selection procedure was monitored by measuring eluted phage titres and by sequencing individual clones after each round of selection.
- Table A shows the successive phage titres for elution in the presence and absence of enzyme after 1, 2 and 3 rounds of selection.
- Clearly, the ratio of specifically eluted phage: non-specifically eluted phage (ie phage eluted with enzyme:phage eluted without enzyme) increases dramatically from
round 1 toround 3, suggesting that the population of good substrates is increasing with each round of selection. - Sequencing of 10 isolates from the starting library showed them all to consist of the wild-type pDM0464 sequence. This is attributed to the fact that after digestion with ApaI, the SalI site is very close to the end of the DNA fragment, thus leading to low efficiency of digestion. Nevertheless, there are only 400 possible sequences in the library, so this population should still be well represented.
- Tables B1 and B2 shows the sequences of isolates obtained after
round 2 andround 3 of selection. After 2 rounds of selection, there is clearly a high incidence of histidine residues. This is exactly what is expected: as described in example XIII, A64SAL subtilisin requires a histidine residue in the substrate as it employs a substrate-assisted catalytic mechanism. After 3 rounds of selection, each of the 10 clones sequenced has a histidine in the randomised cassette. Note, however, that 2 of the sequences are of pDM0411, which was not present in the starting library and is therefore a contaminant.TABLE A Titration of initial phage pools and eluted phage from 3 rounds of selective enrichment Colony forming units (cfu) were estimated by plating out 10 μl of 10-fold dilutions of phage on 10 μl spots of XL-1 blue cells, on LB agar plates containing 50 μg/ ml carbenicillin ROUND 1 Starting library: 3 × 1012 cfu/ml LIBRARY: +500 nM A64SAL 4 × 103 cfu/10 μl no enzyme 3 × 103 cfu/10 μl pDM0411: +500 nM A64SAL 2 × 106 cfu/10 μl (control) no enzyme 8 × 103 cfu/10 μl ROUND 2 Round 1 library:7 × 1012 cfu/ml LIBRARY: +500 nM A64SAL 3 × 104 cfu/10 μl no enzyme 6 × 103 cfu/10 μl pDM0411: +500 nM A64SAL 3 × 106 cfu/10 μl (control) no enzyme 1.6 × 104 cfu/10 μl ROUND 3 Round 2 library:7 × 1011 cfu/ml LIBRARY: +500 nM A64SAL 1 × 105 cfu/10 μl no enzyme <103 cfu/10 μl pDM0411: +500 nM A64SAL 5 × 106 cfu/10 μl (control) no enzyme 3 × 104 cfu/10 μl -
TABLE B1 Sequences of eluted phage after 2 rounds of selective enrichment. No. of Sequence occurrences After round 2: * * A A H Y T R Q (SEQ ID NO:97) . . . GCT GCT CAC TAC ACC CGG CAA . . . (SEQ ID NO:32) 2 A A H M T R Q (SEQ ID NO:98) . . . GCT GCT CAC ATG ACC CGG CAA . . . (SEQ ID NO:33) 1 A A L H T R Q (SEQ ID NO:99) . . . GCT GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO:34) 1 A A L H T R Q (SEQ ID NO:99) . . . GCT GCT CTG CAC ACC CGG CAA . . . (SEQ ID NO:35) 1 A A H T R Q (SEQ ID NO:100) . . . GCT GCT CAC ACC CGG CAA . . . (SEQ ID NO:36) 1# A A ? H T R Q (SEQ ID NO:101) . . . GCT GCT ??? CAC ACC CGG CAA (SEQ ID NO:37) 1## . . . wild- type pDM0454 3
#-spurious deletion of 1 codon within the cassette
##-ambiguous sequence
All protein sequences should be of the form AA*TRQ, where * represents a randomised codon. In the table below, the randomised codons and amino acids are underlined and in bold.
-
TABLE B2 Sequences of eluted phage after 3 rounds of selective enrichment. No. of Sequence occurrences After round 3: * * A A H Y T R Q (SEQ ID NO:97) . . . GCT GCT CAC TAT ACG CGT CAG . . . (SEQ ID NO:38) 2# A A L H T R Q (SEQ ID NO:99) . . . GCT GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO:34) 2 A A Q H T R Q (SEQ ID NO:102) . . . GCT GCT CAG CAC ACC CGG CAA . . . (SEQ ID NO:39) 1 A A T H T R Q (SEQ ID NO:103) . . . GCT GCT ACG CAC ACC CGG CAA . . . (SEQ ID NO:40) 1 A A H S R Q (SEQ ID NO:104) . . . GCT GCT CAC TCC CGG CAA . . . (SEQ ID NO:41) 1 A A H H T R Q (SEQ ID NO:105) . . . GCT GCT CAT CAT ACC CGG CAA (SEQ ID NO:42) 1## A A H F R Q (SEQ ID NO:106) . . . GCT GCT CAC TTC CGG CAA . . . (SEQ ID NO:43) 1 A A H T R Q (SEQ ID NO:100) . . . GCT GCT CAC ACC CGG CAA . . . (SEQ ID NO:36) 1
#-contaminating sequence from pDM0411
##-contains the “illegal” codon CAT - T should not appear in the 3rd position of a codon.
All protein sequences should be of the form AA**TRQ, where * represents a randomised codon. In the table below, the randomised codons and amino acids are underlined and in bold.
-
Claims (52)
1. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising
a transcription regulatory element operably linked to
a gene fusion encoding a fusion protein wherein the gene fusion comprises
a first gene encoding a polypeptide, and
a second gene encoding at least a portion of a phage coat protein;
(b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids;
(c) transforming suitable host cells with the plasmids;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) separating the phagemid particles that bind from those that do not.
2. The method of claim 1 further comprising infecting a suitable host cells with the phagemid particles that bind and repeating steps (d) through (g).
3. The method of claim 2 wherein the steps are repeated one or more times.
4. The method of claim 1 wherein the expression vector further comprises a secretory signal sequence.
5. The method of claim 1 wherein the transcription regulatory element is a promoter system selected from the group; lac Z, pho A, tryptophan, tac, λPL, bacteriophage T7, and combinations thereof.
6. The method of claim 1 wherein the first gene encodes a mammalian protein.
7. The method of claim 6 wherein the protein is selected from the group;
growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β, platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, HER2 ligands, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B, or C, or immuno globulins, and fragments of the above-listed proteins.
8. The method of claim 7 wherein the protein is a human protein.
9. The method of claim 8 wherein the protein comprises more than about 100 amino acid residues.
10. The method of claim 1 wherein the protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target, and the mutations are primarily produced at positions corresponding to codons encoding the amino acids.
11. The method of claim 10 wherein the rigid secondary structures comprise structures selected from the group; α-(3.613)helix, 310 helix, π-(4.416)helix, parallel and anti-parallel β-pleated sheets, reverse turns, and non-ordered structures.
12. The method of claim 10 wherein the mutations are produced at more than one codon.
13. The method of claim 12 wherein the mutations are produced on more than one rigid secondary structure.
14. The method of claim 1 wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174.
15. The method of claim 14 wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein.
16. The method of claim 15 wherein the host is E. coli.
17. The method of claim 16 wherein the plasmid is under tight control of the transcription regulatory element.
18. The method of claim 17 wherein the amount is less than about 1%.
19. The method of claim 18 wherein the amount is less than 20% the amount of phagemid particles displaying a single copy of the fusion protein.
20. The method of claim 19 wherein the amount is less than 10%.
21. The method of claim 1 further comprising in step (a), inserting a DNA triplet, encoding an mRNA suppressible terminator codon between said first gene encoding a polypeptide, and said second gene encoding at least a portion of a phage coat protein.
22. The method of claim 21 wherein said mRNA suppressible terminator codon is selected from the following: UAG (amber), UAA (ocher) and UGA (opel).
23. The method of claim 22 wherein said suppressible mutation results in the detectable production of a fusion polypeptide containing sadi polypeptide and said coat protein when said expression vector is grown in a suppressor host cell; and, when grown in a non-suppressor host cell said polypeptide is synthesized substantially without fusion to said phage coat protein.
24. A human growth hormone variant wherein hGH amino acids 172, 174, 176 and 178 respectively are as a group sequentially selected from one of the following: (1) R,S,F,R; (2) R,A,Y,R; (3) K,T,Y,K; (4) R,S,Y,R; (5) K,A,Y,R; (6) R,F,F,R; (7) K,Q,Y,R; (8) R,T,Y,H; (9) Q,R,Y,R; (10) K,K,Y,K; (11) R,S,F,S; and (12) K,S,N,R.
25. A phagemid comprising a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein, wherein a DNA triplet codon encoding an mRNA suppressible terminator codon selected from UAG, UAA and UGA is inserted between the fused ends of the first and second genes, or is substituted for an amino acid encoding triplet codon adjacent to the gene fusion junction.
26. The phagemid of claim 25 wherein said first gene encodes a mammalian protein.
27. The phagemid of claim 26 wherein the protein is selected from the group: growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β, platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth-I and - II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B or C immuno globulins, and fragments of the above-listed proteins.
28. The phagemid of claim 27 wherein said protein is a human protein.
29. The phagemid of claim 28 wherein the protein comprises more than about 100 amino acid residues.
30. The phagemid of claim 25 wherein said protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target.
31. The phagemid of claim 30 wherein said rigid secondary structures comprises structures selected from the group; α-(3.613)helix, 310 helix, π-(4.416)helix, parallel and anti-parallel β-pleated sheets, reverse turns, and non-ordered structures.
32. The phagemid of claim 25 wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174.
33. The phagemid of claim 32 wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein.
34. The phagemid of claim 33 wherein the host is the E. coli wild type or suppressor type.
35. The phagemid of claim 34 wherein the plasmid is under tight control of the transcription regulatory element.
36. The phagemid of claim 35 wherein the number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particles is less than 1%.
37. The phagemid of claim 36 wherein said number of phagemid particles is less than about 10%.
38. The phagemid of claim 37 wherein the number of phagemid particles is less than about 20%.
39. A human growth variant wherein hGH amino acids 10, 14, 18, and 21 respectively are as a group sequentially selected from one of the following:
(1) H,G,N,N; (2) A,W,D,N; (3) F,S,F,L; (4) Y,T,V,N and (5) I,N,I,N.
40. A human growth variant wherein hGH amino acids 174 is serine and 176 is tyrosine and hGH amino acids 167, 171, 175 and 179 respectively are as a group sequentially selected from one of the following:
(1) N,S,T,T; (2) E,S,T,I; (3) K,S,T,L; (4) N,N,T,T; (5) R,D,I,I; and (6) N,S,T,Q.
41. A method for selecting novel binding polypeptides comprising
(a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein;
(b) mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors;
(c) transforming suitable host cells with the vectors;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) separating the phagemid particles that bind from those that do not.
42. The method of claim 41 wherein the expression vector further comprises a secretory signal sequence operably linked to the DNA encoding each subunit of the protein of interest.
43. The method of claim 42 wherein the protein of interest is a mammalian protein.
44. The method of claim 43 wherein the protein of interest is selected from the group; insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, monoclonal and polyclonal antibodies, lung surfactant, integrin receptors, insulin-like growth factor-I and -II, and fragments of the above-listed proteins.
45. The method of claim 44 wherein the protein of interest is a humanized antibody.
46. The method of claim 45 wherein the protein of interest is a humanized Fab fragment capable of binding to the HER-2 receptor (human epidermal growth factor receptor-2).
47. A human growth hormone (hGH) variant wherein hGH amino acid glutamate174 is replaced by serine174 and phenylalanine176 is replaced by tyrosine176 and one or more of the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I 179 are replaced by another natural amino acid.
48. The hGH variant of claim 47 wherein the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I 179 respectively are as a group replaced with a corresponding amino acid sequentially selected from one of the following groups:
49. The method of claim 48 wherein said human growth hormone variant (11) further contains leucine15 replaced by arginine15 and lysine168 replaced by arginine168.
50. The method of claim 48 wherein said human growth hormone variant (40) further contains phenylalanine176
51. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising
a transcription regulatory element operably linked to
a gene fusion encoding a fusion protein wherein the gene fusion comprises
a first gene encoding a polypeptide operable connected to a linking amino acid sequence, and
a second gene encoding at least a portion of a phage coat protein;
(b) mutating the vector at one or more selected positions within the amino acid linking sequence of the first gene thereby forming a family of related plasmids;
(c) transforming suitable host cells with the plasmids;
(d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;
(e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;
(f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and
(g) contacting the bound phagemid particles with a protease capable of hydrolysing the linking a amino acid sequence of at least a portion of the bound phagmid particles, and
(h) isolating the hydrolyzed phagmid particles.
52. The method of claim 51 further comprising infecting suitable host cells with the hydrolyzed phagemid particles and repeating steps (d) through (h).
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/761,180 US20080038717A1 (en) | 1990-12-03 | 2007-06-11 | Enrichment method for variant proteins with altered binding properties |
US12/508,859 US20100035236A1 (en) | 1990-12-03 | 2009-07-24 | Enrichment method for variant proteins with altered binding properties |
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62166790A | 1990-12-03 | 1990-12-03 | |
US68340091A | 1991-04-10 | 1991-04-10 | |
US71530091A | 1991-06-14 | 1991-06-14 | |
US74361491A | 1991-08-09 | 1991-08-09 | |
US08/050,058 US5750373A (en) | 1990-12-03 | 1991-12-03 | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
PCT/US1991/009133 WO1992009690A2 (en) | 1990-12-03 | 1991-12-03 | Enrichment method for variant proteins with altered binding properties |
US08/463,587 US5821047A (en) | 1990-12-03 | 1995-06-05 | Monovalent phage display |
US92234597A | 1997-09-03 | 1997-09-03 | |
US71764100A | 2000-11-21 | 2000-11-21 | |
US11/199,062 US20060115874A1 (en) | 1990-12-03 | 2005-08-08 | Enrichment method for variant proteins with altered binding properties |
US11/761,180 US20080038717A1 (en) | 1990-12-03 | 2007-06-11 | Enrichment method for variant proteins with altered binding properties |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/199,062 Continuation US20060115874A1 (en) | 1990-12-03 | 2005-08-08 | Enrichment method for variant proteins with altered binding properties |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/508,859 Continuation US20100035236A1 (en) | 1990-12-03 | 2009-07-24 | Enrichment method for variant proteins with altered binding properties |
Publications (1)
Publication Number | Publication Date |
---|---|
US20080038717A1 true US20080038717A1 (en) | 2008-02-14 |
Family
ID=27505166
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/050,058 Expired - Lifetime US5750373A (en) | 1988-10-28 | 1991-12-03 | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US08/463,587 Expired - Lifetime US5821047A (en) | 1990-12-03 | 1995-06-05 | Monovalent phage display |
US08/463,667 Expired - Lifetime US5834598A (en) | 1990-12-03 | 1995-06-05 | Human growth hormone variants |
US08/923,854 Expired - Fee Related US6040136A (en) | 1990-12-03 | 1997-09-03 | Enrichment method for variant proteins with altered binding properties |
US11/199,062 Abandoned US20060115874A1 (en) | 1990-12-03 | 2005-08-08 | Enrichment method for variant proteins with altered binding properties |
US11/761,180 Abandoned US20080038717A1 (en) | 1990-12-03 | 2007-06-11 | Enrichment method for variant proteins with altered binding properties |
US12/508,859 Abandoned US20100035236A1 (en) | 1990-12-03 | 2009-07-24 | Enrichment method for variant proteins with altered binding properties |
Family Applications Before (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/050,058 Expired - Lifetime US5750373A (en) | 1988-10-28 | 1991-12-03 | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US08/463,587 Expired - Lifetime US5821047A (en) | 1990-12-03 | 1995-06-05 | Monovalent phage display |
US08/463,667 Expired - Lifetime US5834598A (en) | 1990-12-03 | 1995-06-05 | Human growth hormone variants |
US08/923,854 Expired - Fee Related US6040136A (en) | 1990-12-03 | 1997-09-03 | Enrichment method for variant proteins with altered binding properties |
US11/199,062 Abandoned US20060115874A1 (en) | 1990-12-03 | 2005-08-08 | Enrichment method for variant proteins with altered binding properties |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/508,859 Abandoned US20100035236A1 (en) | 1990-12-03 | 2009-07-24 | Enrichment method for variant proteins with altered binding properties |
Country Status (9)
Country | Link |
---|---|
US (7) | US5750373A (en) |
EP (1) | EP0564531B1 (en) |
AT (1) | ATE164395T1 (en) |
CA (2) | CA2405246A1 (en) |
DE (1) | DE69129154T2 (en) |
DK (1) | DK0564531T3 (en) |
ES (1) | ES2113940T3 (en) |
GR (1) | GR3026468T3 (en) |
WO (1) | WO1992009690A2 (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070237764A1 (en) * | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US20080194477A1 (en) * | 2002-09-09 | 2008-08-14 | Rene Gantier | Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules |
US7884073B2 (en) | 2004-11-04 | 2011-02-08 | Hanall Biopharma Co., Ltd. | Modified growth hormone |
US20110177095A1 (en) * | 2009-12-16 | 2011-07-21 | Abbott Biotherapeutics Corporation | Anti-her2 antibodies and their uses |
US8679490B2 (en) | 2005-11-07 | 2014-03-25 | Genentech, Inc. | Binding polypeptides with diversified and consensus VH/VL hypervariable sequences |
US8911734B2 (en) | 2010-12-01 | 2014-12-16 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9067988B2 (en) | 2010-12-01 | 2015-06-30 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies |
US9078878B2 (en) | 2010-12-01 | 2015-07-14 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9539324B2 (en) | 2010-12-01 | 2017-01-10 | Alderbio Holdings, Llc | Methods of preventing inflammation and treating pain using anti-NGF compositions |
US9884909B2 (en) | 2010-12-01 | 2018-02-06 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
US11214610B2 (en) | 2010-12-01 | 2022-01-04 | H. Lundbeck A/S | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris |
Families Citing this family (2190)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5723286A (en) * | 1990-06-20 | 1998-03-03 | Affymax Technologies N.V. | Peptide library and screening systems |
US6916605B1 (en) | 1990-07-10 | 2005-07-12 | Medical Research Council | Methods for producing members of specific binding pairs |
GB9206318D0 (en) * | 1992-03-24 | 1992-05-06 | Cambridge Antibody Tech | Binding substances |
GB9015198D0 (en) | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
US6172197B1 (en) | 1991-07-10 | 2001-01-09 | Medical Research Council | Methods for producing members of specific binding pairs |
US7063943B1 (en) | 1990-07-10 | 2006-06-20 | Cambridge Antibody Technology | Methods for producing members of specific binding pairs |
ATE164395T1 (en) * | 1990-12-03 | 1998-04-15 | Genentech Inc | METHOD FOR ENRICHMENT OF PROTEIN VARIANTS WITH MODIFIED BINDING PROPERTIES |
WO1992018619A1 (en) | 1991-04-10 | 1992-10-29 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
WO1992021029A1 (en) | 1991-05-10 | 1992-11-26 | Genentech, Inc. | Selecting ligand agonists and antagonists |
US5858657A (en) * | 1992-05-15 | 1999-01-12 | Medical Research Council | Methods for producing members of specific binding pairs |
US6492160B1 (en) | 1991-05-15 | 2002-12-10 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
DE69230142T2 (en) | 1991-05-15 | 2000-03-09 | Cambridge Antibody Tech | METHOD FOR PRODUCING SPECIFIC BINDING PAIRS |
US5962255A (en) * | 1992-03-24 | 1999-10-05 | Cambridge Antibody Technology Limited | Methods for producing recombinant vectors |
US6225447B1 (en) | 1991-05-15 | 2001-05-01 | Cambridge Antibody Technology Ltd. | Methods for producing members of specific binding pairs |
JP4124480B2 (en) | 1991-06-14 | 2008-07-23 | ジェネンテック・インコーポレーテッド | Immunoglobulin variants |
US6800738B1 (en) * | 1991-06-14 | 2004-10-05 | Genentech, Inc. | Method for making humanized antibodies |
DE4122599C2 (en) | 1991-07-08 | 1993-11-11 | Deutsches Krebsforsch | Phagemid for screening antibodies |
US5270170A (en) * | 1991-10-16 | 1993-12-14 | Affymax Technologies N.V. | Peptide library and screening method |
US5733731A (en) * | 1991-10-16 | 1998-03-31 | Affymax Technologies N.V. | Peptide library and screening method |
PT1696031E (en) | 1991-12-02 | 2010-06-25 | Medical Res Council | Production of anti-self antibodies from antibody segment repertoires and displayed on phage |
US5733743A (en) * | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
US5849535A (en) * | 1995-09-21 | 1998-12-15 | Genentech, Inc. | Human growth hormone variants |
US6043212A (en) | 1993-07-26 | 2000-03-28 | Cor Therapeutics, Inc. | Recombinant C140 receptor, its agonists and antagonists, and nucleic acids encoding the receptor |
NZ278765A (en) | 1993-12-30 | 1998-05-27 | Harvard College | Vertebrate proteins involved in spatial arrangements of differentiated tissues called hedgehog proteins, their production and use |
US6335160B1 (en) | 1995-02-17 | 2002-01-01 | Maxygen, Inc. | Methods and compositions for polypeptide engineering |
US5837458A (en) * | 1994-02-17 | 1998-11-17 | Maxygen, Inc. | Methods and compositions for cellular and metabolic engineering |
US6406855B1 (en) | 1994-02-17 | 2002-06-18 | Maxygen, Inc. | Methods and compositions for polypeptide engineering |
US6576236B1 (en) | 1994-07-01 | 2003-06-10 | Dana Farber Cancer Institute | Methods for stimulating T cell responses by manipulating a common cytokine receptor γ chain |
US7820798B2 (en) * | 1994-11-07 | 2010-10-26 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US7597886B2 (en) * | 1994-11-07 | 2009-10-06 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US6335319B1 (en) * | 1994-11-15 | 2002-01-01 | Metabolic Pharmaceuticals, Inc. | Treatment of obesity |
US7429646B1 (en) | 1995-06-05 | 2008-09-30 | Human Genome Sciences, Inc. | Antibodies to human tumor necrosis factor receptor-like 2 |
US6475806B1 (en) | 1995-06-07 | 2002-11-05 | Praecis Pharmaceuticals, Inc. | Anchor libraries and identification of peptide binding sequences |
US7368111B2 (en) | 1995-10-06 | 2008-05-06 | Cambridge Antibody Technology Limited | Human antibodies specific for TGFβ2 |
DK0859959T3 (en) * | 1995-11-10 | 2003-11-24 | Elan Corp Plc | Peptides that enhance tissue transport and methods for identifying and using them |
US6090382A (en) | 1996-02-09 | 2000-07-18 | Basf Aktiengesellschaft | Human antibodies that bind human TNFα |
US7888466B2 (en) | 1996-01-11 | 2011-02-15 | Human Genome Sciences, Inc. | Human G-protein chemokine receptor HSATU68 |
WO1997035194A2 (en) * | 1996-03-21 | 1997-09-25 | President And Fellows Of Harvard College | Enantiomeric screening process, and compositions therefor |
US6777217B1 (en) | 1996-03-26 | 2004-08-17 | President And Fellows Of Harvard College | Histone deacetylases, and uses related thereto |
US6361938B1 (en) | 1996-11-08 | 2002-03-26 | Elan Corporation, Plc | Peptides which enhance transport across tissues and methods of identifying and using the same |
US20070185032A1 (en) * | 1996-12-11 | 2007-08-09 | Praecis Pharmaceuticals, Inc. | Pharmaceutical formulations for sustained drug delivery |
US6497874B1 (en) | 1997-02-05 | 2002-12-24 | Maardh Sven | Recombinant phages |
JP2001524824A (en) | 1997-04-16 | 2001-12-04 | ミレニアム・フアーマシユーチカルズ・インコーポレーテツド | CRSP proteins (secretory proteins rich in cysteine), nucleic acid molecules encoding them and uses thereof |
US6306826B1 (en) | 1997-06-04 | 2001-10-23 | The Regents Of The University Of California | Treatment of heart failure with growth hormone |
US6172213B1 (en) * | 1997-07-02 | 2001-01-09 | Genentech, Inc. | Anti-IgE antibodies and method of improving polypeptides |
US5994511A (en) * | 1997-07-02 | 1999-11-30 | Genentech, Inc. | Anti-IgE antibodies and methods of improving polypeptides |
US6639050B1 (en) | 1997-07-21 | 2003-10-28 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
US7378506B2 (en) | 1997-07-21 | 2008-05-27 | Ohio University | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins |
WO1999006587A2 (en) | 1997-08-01 | 1999-02-11 | Morphosys Ag | Novel method and phage for the identification of nucleic acid sequences encoding members of a multimeric (poly)peptide complex |
GB2375765B (en) * | 1997-11-14 | 2003-02-26 | Kymed Gb Ltd | Tagged growth hormone molecules |
GB9723955D0 (en) | 1997-11-14 | 1998-01-07 | Generic Biolog Limited | Improvements in or relating to detection of molecules in samples |
US7192589B2 (en) | 1998-09-16 | 2007-03-20 | Genentech, Inc. | Treatment of inflammatory disorders with STIgMA immunoadhesins |
EP1481990B1 (en) | 1997-11-21 | 2007-06-06 | Genentech, Inc. | A-33 related antigens and their pharmacological uses |
CA2323776C (en) | 1998-03-19 | 2010-04-27 | Human Genome Sciences, Inc. | Cytokine receptor common gamma chain like |
US7163682B2 (en) | 1998-04-13 | 2007-01-16 | The Forsyth Institute | Glucan binding protein and glucosyltransferase immunogens |
US7056517B2 (en) | 1998-04-13 | 2006-06-06 | The Forsyth Institute | Glucosyltransferase immunogens |
EP1100890A2 (en) | 1998-07-27 | 2001-05-23 | Genentech, Inc. | Improved transformation efficiency in phage display through modification of a coat protein |
US6387888B1 (en) * | 1998-09-30 | 2002-05-14 | American Foundation For Biological Research, Inc. | Immunotherapy of cancer through expression of truncated tumor or tumor-associated antigen |
US6485972B1 (en) | 1998-10-15 | 2002-11-26 | President And Fellows Of Harvard College | WNT signalling in reproductive organs |
US6420110B1 (en) | 1998-10-19 | 2002-07-16 | Gpc Biotech, Inc. | Methods and reagents for isolating biologically active peptides |
ATE406907T1 (en) * | 1998-10-28 | 2008-09-15 | Cornell Res Foundation Inc | METHODS FOR REGULATION OF ANGIOGENESIS AND VASCULAR INTEGRITY USING TRK RECEPTOR LIGANDS BDNF, NT-3 AND NT-4 |
US6927024B2 (en) | 1998-11-30 | 2005-08-09 | Genentech, Inc. | PCR assay |
US6696063B1 (en) | 1998-12-30 | 2004-02-24 | Applied Research Systems Ars Holding N.V. | Treatment of HIV-associated dysmorphia/dysmetabolic syndrome (HADDS) with or without lipodystrophy |
US6602705B1 (en) * | 1998-12-31 | 2003-08-05 | Chiron Corporation | Expression of HIV polypeptides and production of virus-like particles |
WO2000039304A2 (en) * | 1998-12-31 | 2000-07-06 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
US7935805B1 (en) | 1998-12-31 | 2011-05-03 | Novartis Vaccines & Diagnostics, Inc | Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof |
AU2596600A (en) | 1998-12-31 | 2000-07-31 | Chiron Corporation | Modified hiv env polypeptides |
DK1141014T3 (en) | 1999-01-06 | 2005-04-11 | Genentech Inc | Insulin-like growth factor (IGF) in mutant variant |
AU762047B2 (en) * | 1999-01-06 | 2003-06-19 | Genentech Inc. | Insulin-like growth factor (IGF) I mutant variants |
EP1179000A4 (en) | 1999-02-26 | 2005-10-12 | Millennium Pharm Inc | Secreted proteins and uses thereof |
EP1161451A4 (en) | 1999-02-26 | 2006-05-17 | Human Genome Sciences Inc | Human endokine alpha and methods of use |
AU767066B2 (en) | 1999-03-03 | 2003-10-30 | Curis, Inc. | Methods of modulating lipid metabolism and storage |
TR200501367T2 (en) | 1999-03-25 | 2005-09-21 | Abbott Gmbh & Co. Kg | Human antibodies that bind human IL-12 and methods for producing them. |
US6914128B1 (en) | 1999-03-25 | 2005-07-05 | Abbott Gmbh & Co. Kg | Human antibodies that bind human IL-12 and methods for producing |
US7883704B2 (en) | 1999-03-25 | 2011-02-08 | Abbott Gmbh & Co. Kg | Methods for inhibiting the activity of the P40 subunit of human IL-12 |
AU4057600A (en) * | 1999-03-31 | 2000-10-16 | Rosetta Inpharmatics, Inc. | Methods for the identification of reporter and target molecules using comprehensive gene expression profiles |
DE19915057A1 (en) | 1999-04-01 | 2000-10-19 | Forschungszentrum Borstel | Monoclonal antibodies to the human Mcm3 protein, process for their preparation and their use |
US6492497B1 (en) | 1999-04-30 | 2002-12-10 | Cambridge Antibody Technology Limited | Specific binding members for TGFbeta1 |
AU771534B2 (en) | 1999-05-05 | 2004-03-25 | Phylogica Limited | Isolating biological modulators from biodiverse gene fragment libraries |
US7803765B2 (en) * | 1999-05-05 | 2010-09-28 | Phylogica Limited | Methods of constructing biodiverse gene fragment libraries and biological modulators isolated therefrom |
US7270969B2 (en) * | 1999-05-05 | 2007-09-18 | Phylogica Limited | Methods of constructing and screening diverse expression libraries |
US6197517B1 (en) | 1999-05-21 | 2001-03-06 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US7396905B1 (en) | 1999-05-21 | 2008-07-08 | Mckeon Frank | Calcipressins: endogenous inhibitors of calcineurin, uses and reagents related thereto |
US6200803B1 (en) | 1999-05-21 | 2001-03-13 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US6221597B1 (en) | 1999-05-21 | 2001-04-24 | Rosetta Inpharmatics, Inc. | Essential genes of yeast as targets for antifungal agents, herbicides, insecticides and anti-proliferative drugs |
US20030166003A1 (en) * | 1999-06-14 | 2003-09-04 | Cochran Andrea G. | Structured peptide scaffold for displaying turn libraries on phage |
ES2242620T3 (en) | 1999-06-14 | 2005-11-16 | Genentech, Inc. | PEPTIDIC STRUCTURES TO DISPLAY LIBRARIES OF TURNS OF A PAYMENT. |
US7291714B1 (en) | 1999-06-30 | 2007-11-06 | Millennium Pharmaceuticals, Inc. | Glycoprotein VI and uses thereof |
US20040001826A1 (en) | 1999-06-30 | 2004-01-01 | Millennium Pharmaceuticals, Inc. | Glycoprotein VI and uses thereof |
DK1144607T5 (en) | 1999-07-20 | 2009-10-05 | Morphosys Ag | Process for presenting (poly) peptides / proteins on bacteriophage particles via disulfide bonds |
WO2001014557A1 (en) | 1999-08-23 | 2001-03-01 | Dana-Farber Cancer Institute, Inc. | Pd-1, a receptor for b7-4, and uses therefor |
DE19943743C2 (en) * | 1999-09-03 | 2002-02-07 | Jerini Biotools Gmbh | Procedure for the identification of binding partners with position-specific arrays |
EP1222209A2 (en) * | 1999-10-20 | 2002-07-17 | Genentech, Inc. | Modulation of t cell differentiation for the treatment of t helper cell mediated diseases |
US20060073509A1 (en) * | 1999-11-18 | 2006-04-06 | Michael Kilpatrick | Method for detecting and quantitating multiple subcellular components |
US8168178B2 (en) | 1999-11-30 | 2012-05-01 | Curis, Inc. | Methods and compositions for regulating lymphocyte activity |
US6951839B1 (en) | 1999-11-30 | 2005-10-04 | Curis, Inc. | Methods and compositions for regulating lymphocyte activity |
CA2393869A1 (en) * | 1999-12-15 | 2001-06-21 | Genetech,Inc. | Shotgun scanning, a combinatorial method for mapping functional protein epitopes |
US20020004247A1 (en) * | 1999-12-23 | 2002-01-10 | Genentech, Inc. | Assay method |
AU784883B2 (en) | 1999-12-30 | 2006-07-20 | President And Fellows Of Harvard College | Methods and compositions relating to modulation of hepatocyte growth, plasma cell differentiation or T cell subset activity by modulation of XBP-1 activity |
EP2332579A3 (en) | 2000-02-10 | 2011-09-21 | Abbott Laboratories | Antibodies that bind human interleukin-18 and methods of making and using |
US6632645B1 (en) | 2000-03-02 | 2003-10-14 | Promega Corporation | Thermophilic DNA polymerases from Thermoactinomyces vulgaris |
US6436677B1 (en) | 2000-03-02 | 2002-08-20 | Promega Corporation | Method of reverse transcription |
US20030129724A1 (en) | 2000-03-03 | 2003-07-10 | Grozinger Christina M. | Class II human histone deacetylases, and uses related thereto |
DK2857516T3 (en) | 2000-04-11 | 2017-08-07 | Genentech Inc | Multivalent antibodies and uses thereof |
CA2405709A1 (en) | 2000-04-12 | 2001-10-25 | Human Genome Sciences, Inc. | Albumin fusion proteins |
EP2199393B1 (en) * | 2000-04-17 | 2012-10-31 | Dyax Corp. | Methods of constructing display libraries of genetic packages for members of a diverse family of peptides |
US8288322B2 (en) | 2000-04-17 | 2012-10-16 | Dyax Corp. | Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries |
DE60118935D1 (en) * | 2000-04-24 | 2006-05-24 | Univ Yale New Haven | DNA & PROTEIN BINDING MINIATURE PROTEINS |
US7495070B2 (en) * | 2000-04-24 | 2009-02-24 | Yale University | Protein binding miniature proteins |
ES2301547T3 (en) | 2000-05-16 | 2008-07-01 | Genentech, Inc. | TREATMENT OF CARTILAGO DISORDERS. |
US6573370B1 (en) | 2000-05-19 | 2003-06-03 | Regents Of The University Of Michigan | PON3 and uses thereof |
EP1714661A3 (en) | 2000-05-19 | 2012-03-14 | The Center for Blood Research, INC. | Methods for diagnosing and treating hemostatic disorders by modulating p-selectin activity |
US20030031675A1 (en) | 2000-06-06 | 2003-02-13 | Mikesell Glen E. | B7-related nucleic acids and polypeptides useful for immunomodulation |
ES2343351T3 (en) | 2000-06-08 | 2010-07-29 | Immune Disease Institute, Inc. | PROCEDURES AND COMPOSITIONS TO INHIBIT THE INJURY BY IMMUNOGLOBULIN REPERFUSION. |
AU2001282856A1 (en) | 2000-06-15 | 2001-12-24 | Human Genome Sciences, Inc. | Human tumor necrosis factor delta and epsilon |
PT2281843T (en) | 2000-06-16 | 2017-01-02 | Human Genome Sciences Inc | Antibodies that immunospecifically bind to blys |
WO2002000617A2 (en) * | 2000-06-28 | 2002-01-03 | Bristol-Myers Squibb Company | Selective androgen receptor modulators and methods for their identification, design and use |
ES2402546T3 (en) | 2000-06-28 | 2013-05-06 | Genetics Institute, Llc | PD-L2 molecules: new PD-1 ligands and their uses |
IL153567A0 (en) | 2000-06-29 | 2003-07-06 | Abbott Lab | Dual specificity antibodies and methods of making and using |
US6951947B2 (en) * | 2000-07-13 | 2005-10-04 | The Scripps Research Institute | Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation |
US7176037B2 (en) * | 2000-07-13 | 2007-02-13 | The Scripps Research Institute | Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation |
EP1301532B1 (en) | 2000-07-14 | 2011-02-02 | CropDesign N.V. | Plant cyclin-dependent kinase inhibitors |
US6878861B2 (en) | 2000-07-21 | 2005-04-12 | Washington State University Research Foundation | Acyl coenzyme A thioesterases |
CA2415473A1 (en) | 2000-07-27 | 2002-02-07 | Genentech, Inc. | Apo-2l receptor agonist and cpt-11 synergism |
US20080194022A1 (en) * | 2000-08-03 | 2008-08-14 | Clarke Michael F | Isolation and use of solid tumor stem cells |
US8044259B2 (en) | 2000-08-03 | 2011-10-25 | The Regents Of The University Of Michigan | Determining the capability of a test compound to affect solid tumor stem cells |
US6984522B2 (en) | 2000-08-03 | 2006-01-10 | Regents Of The University Of Michigan | Isolation and use of solid tumor stem cells |
UA81743C2 (en) | 2000-08-07 | 2008-02-11 | Центокор, Инк. | HUMAN MONOCLONAL ANTIBODY WHICH SPECIFICALLY BINDS TUMOR NECROSIS FACTOR ALFA (TNFα), PHARMACEUTICAL MIXTURE CONTAINING THEREOF, AND METHOD FOR TREATING ARTHRITIS |
TWI283182B (en) * | 2000-08-07 | 2007-07-01 | Nektar Therapeutics | Inhalable spray dried 4-helix bundle protein powders having minimized aggregation |
US6902734B2 (en) | 2000-08-07 | 2005-06-07 | Centocor, Inc. | Anti-IL-12 antibodies and compositions thereof |
US7288390B2 (en) | 2000-08-07 | 2007-10-30 | Centocor, Inc. | Anti-dual integrin antibodies, compositions, methods and uses |
ATE552859T1 (en) * | 2000-09-13 | 2012-04-15 | Praecis Pharm Inc | PHARMACEUTICAL FORMULATIONS FOR CONTINUOUS DELIVERY OF PEPTIDES |
GB0022978D0 (en) | 2000-09-19 | 2000-11-01 | Oxford Glycosciences Uk Ltd | Detection of peptides |
DE60135158D1 (en) | 2000-09-26 | 2008-09-11 | Genentech Inc | ANTAGONISTS OF THE IGE RECEPTOR |
US7393532B1 (en) | 2000-10-18 | 2008-07-01 | Genentech, Inc. | Modulation of T cell differentiation for the treatment of T helper cell mediated diseases |
US6673580B2 (en) * | 2000-10-27 | 2004-01-06 | Genentech, Inc. | Identification and modification of immunodominant epitopes in polypeptides |
US6841359B2 (en) * | 2000-10-31 | 2005-01-11 | The General Hospital Corporation | Streptavidin-binding peptides and uses thereof |
AU4155602A (en) | 2000-11-01 | 2002-06-18 | Elusys Therapeutics Inc | Method of producing biospecific molecules by protein trans-splicing |
NZ526617A (en) | 2000-11-28 | 2004-09-24 | Wyeth Corp | Expression analysis of KIAA nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
EP2338512A1 (en) | 2000-11-28 | 2011-06-29 | MedImmune, LLC | Methods of administering/dosing anti-RSV antibodies for prophylaxis and treatment |
PL365605A1 (en) | 2000-11-28 | 2005-01-10 | Wyeth | Expression analysis of fkbp nucleic acids and polypeptides useful in the diagnosis and treatment of prostate cancer |
US20040253242A1 (en) * | 2000-12-05 | 2004-12-16 | Bowdish Katherine S. | Rationally designed antibodies |
US7396917B2 (en) * | 2000-12-05 | 2008-07-08 | Alexion Pharmaceuticals, Inc. | Rationally designed antibodies |
CA2436671C (en) | 2000-12-05 | 2015-02-03 | Alexion Pharmaceuticals, Inc. | Rationally designed antibodies |
US20060057651A1 (en) * | 2000-12-08 | 2006-03-16 | Bowdish Katherine S | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
WO2002059280A2 (en) * | 2000-12-08 | 2002-08-01 | Alexion Pharmaceuticals, Inc. | Chronic lymphocytic leukemia cell line and its use for producing an antibody |
US7408041B2 (en) * | 2000-12-08 | 2008-08-05 | Alexion Pharmaceuticals, Inc. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
US9249229B2 (en) * | 2000-12-08 | 2016-02-02 | Alexion Pharmaceuticals, Inc. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
US20040198661A1 (en) * | 2000-12-08 | 2004-10-07 | Bowdish Katherine S. | Polypeptides and antibodies derived from chronic lymphocytic leukemia cells and uses thereof |
EP1355919B1 (en) | 2000-12-12 | 2010-11-24 | MedImmune, LLC | Molecules with extended half-lives, compositions and uses thereof |
ATE498718T1 (en) | 2000-12-18 | 2011-03-15 | Dyax Corp | DIRECTED LIBRARIES THAT ARE GENETICALLY PACKAGED |
WO2002051870A2 (en) * | 2000-12-22 | 2002-07-04 | GRAD, Carole Legal Representative of KAPLAN, Howard | Phage display libraries of human vh fragments |
ES2390425T3 (en) | 2000-12-22 | 2012-11-12 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Use of repulsive targeting molecules (RGM) and their modulators |
WO2002053596A2 (en) | 2001-01-05 | 2002-07-11 | Pfizer Inc. | Antibodies to insulin-like growth factor i receptor |
CN1564826A (en) | 2001-02-09 | 2005-01-12 | 人类基因组科学公司 | Human G-protein chemokine receptor (CCR5) HDGNR10 |
US7087726B2 (en) | 2001-02-22 | 2006-08-08 | Genentech, Inc. | Anti-interferon-α antibodies |
NZ537401A (en) | 2001-02-23 | 2006-08-31 | Dsm Ip Assets B | Proteases from Aspergillus niger |
US20030068320A1 (en) * | 2001-03-02 | 2003-04-10 | Christine Dingivan | Methods of administering/dosing CD2 antagonists for the prevention and treatment of autoimmune disorders or inflammatory disorders |
EP1975620A3 (en) | 2001-03-02 | 2008-12-24 | GPC Biotech AG | Three hybrid assay system |
US8231878B2 (en) * | 2001-03-20 | 2012-07-31 | Cosmo Research & Development S.P.A. | Receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
US20090081199A1 (en) * | 2001-03-20 | 2009-03-26 | Bioxell S.P.A. | Novel receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
US8981061B2 (en) | 2001-03-20 | 2015-03-17 | Novo Nordisk A/S | Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof |
CA2342376C (en) * | 2001-03-20 | 2013-11-12 | Marco Colonna | A receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
MXPA03008454A (en) | 2001-03-22 | 2004-06-30 | Abbott Gmbh & Co Kg | Transgenic animals expressing antibodies specific for genes of interest and uses thereof. |
MXPA03008959A (en) | 2001-04-02 | 2004-10-15 | Wyeth Corp | Pd-1, a receptor for b7-4, and uses therefor. |
KR20030093316A (en) | 2001-04-13 | 2003-12-06 | 휴먼 게놈 사이언시즈, 인코포레이티드 | Vascular endothelial growth factor 2 |
CA2444133A1 (en) | 2001-04-16 | 2002-10-24 | Wyeth Holdings Corporation | Novel streptococcus pneumoniae open reading frames encoding polypeptide antigens and uses thereof |
US6914123B2 (en) * | 2001-04-17 | 2005-07-05 | Genentech, Inc. | Hairpin peptides with a novel structural motif and methods relating thereto |
US7244853B2 (en) | 2001-05-09 | 2007-07-17 | President And Fellows Of Harvard College | Dioxanes and uses thereof |
AU2002309647C1 (en) | 2001-05-25 | 2008-09-11 | Human Genome Sciences, Inc. | Antibodies that immunospecifically bind to trail receptors |
CA2469843A1 (en) * | 2001-06-04 | 2002-12-12 | Ikonisys Inc. | Method for detecting infectious agents using computer controlled automated image analysis |
US20030148380A1 (en) * | 2001-06-05 | 2003-08-07 | Belcher Angela M. | Molecular recognition of materials |
US20030113714A1 (en) * | 2001-09-28 | 2003-06-19 | Belcher Angela M. | Biological control of nanoparticles |
US20050164515A9 (en) * | 2001-06-05 | 2005-07-28 | Belcher Angela M. | Biological control of nanoparticle nucleation, shape and crystal phase |
US20070160576A1 (en) | 2001-06-05 | 2007-07-12 | Genentech, Inc. | IL-17A/F heterologous polypeptides and therapeutic uses thereof |
CA2817619A1 (en) | 2001-06-08 | 2002-12-08 | Abbott Laboratories (Bermuda) Ltd. | Methods of administering anti-tnf.alpha. antibodies |
ES2372321T3 (en) | 2001-06-20 | 2012-01-18 | Genentech, Inc. | COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF A LUNG TUMOR. |
US20050107595A1 (en) * | 2001-06-20 | 2005-05-19 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
US7803915B2 (en) * | 2001-06-20 | 2010-09-28 | Genentech, Inc. | Antibody compositions for the diagnosis and treatment of tumor |
EP2270187A3 (en) | 2001-06-22 | 2011-08-31 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US20030198621A1 (en) * | 2001-07-05 | 2003-10-23 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B and/or type C polypeptides, polypeptides and uses thereof |
US7211659B2 (en) | 2001-07-05 | 2007-05-01 | Chiron Corporation | Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof |
DE10135039C1 (en) * | 2001-07-18 | 2003-03-13 | Nemod Immuntherapie Ag | Method for isolating large variances of specific molecules for a target molecule from phagemid gene libraries |
US6867189B2 (en) * | 2001-07-26 | 2005-03-15 | Genset S.A. | Use of adipsin/complement factor D in the treatment of metabolic related disorders |
KR100458083B1 (en) * | 2001-08-29 | 2004-11-18 | 주식회사 아이지세라피 | Method for the construction of phage display library using helper phage variants |
AU2002316578A1 (en) * | 2001-08-31 | 2003-03-18 | Chiron Corporation | Polynucleotides encoding antigenic hiv type b polypeptides, polypeptides and uses thereof |
US20030170614A1 (en) * | 2001-08-31 | 2003-09-11 | Megede Jan Zur | Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof |
US20040142325A1 (en) | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
US20030091593A1 (en) * | 2001-09-14 | 2003-05-15 | Cytos Biotechnology Ag | In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles |
KR101008758B1 (en) | 2001-09-18 | 2011-01-14 | 제넨테크, 인크. | Compositions and Methods for the Diagnosis and Treatment of Tumor |
US20030073104A1 (en) * | 2001-10-02 | 2003-04-17 | Belcher Angela M. | Nanoscaling ordering of hybrid materials using genetically engineered mesoscale virus |
US20050123925A1 (en) | 2002-11-15 | 2005-06-09 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
AR039067A1 (en) | 2001-11-09 | 2005-02-09 | Pfizer Prod Inc | ANTIBODIES FOR CD40 |
US7858297B2 (en) | 2001-12-18 | 2010-12-28 | Centre National De La Recherche Scientifique Cnrs | Chemokine-binding protein and methods of use |
EP1458754B1 (en) | 2001-12-18 | 2009-12-09 | Endocube SAS | Novel death associated proteins of the thap family and related par4 pathways involved in apoptosis control |
EP1463807A4 (en) | 2001-12-19 | 2006-04-12 | Bristol Myers Squibb Co | Pichia pastoris formate dehydrogenase and uses therefor |
CA2471363C (en) | 2001-12-21 | 2014-02-11 | Human Genome Sciences, Inc. | Albumin fusion proteins |
KR100623128B1 (en) | 2002-01-02 | 2006-09-14 | 제넨테크, 인크. | Compositions and Methods for the Diagnosis and Treatment of Tumor |
EP1461001A4 (en) * | 2002-01-03 | 2010-03-31 | Scripps Research Inst | Cancer-associated epitope |
US20070166704A1 (en) * | 2002-01-18 | 2007-07-19 | Fei Huang | Identification of polynucleotides and polypeptide for predicting activity of compounds that interact with protein tyrosine kinases and/or protein tyrosine kinase pathways |
US7094579B2 (en) | 2002-02-13 | 2006-08-22 | Xoma Technology Ltd. | Eukaryotic signal sequences for prokaryotic expression |
DE60326214D1 (en) * | 2002-03-01 | 2009-04-02 | Siemens Healthcare Diagnostics | ASSAYS FOR THE MONITORING OF CANCER PATIENTS BASED ON THE MIRRORS OF ANALYTE COMPONENTS OF THE PLASMINOGEN ACTIVATOR SYSTEM IN SAMPLES FROM BODY FLUIDS |
ATE441107T1 (en) | 2002-03-01 | 2009-09-15 | Siemens Healthcare Diagnostics | ASSAY FOR MONITORING CANCER PATIENTS BASED ON LEVELS OF THE EXTRACELLULAR DOMAIN (ECD) ANALYTE OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR), ALONE OR IN COMBINATION WITH OTHER ANALYTES, IN BODY FLUID SAMPLES |
AU2003217976A1 (en) | 2002-03-07 | 2003-09-22 | The Forsyth Institute | Immunogenicity of glucan binding protein |
WO2003085093A2 (en) * | 2002-04-01 | 2003-10-16 | Human Genome Sciences, Inc. | Antibodies that specifically bind to gmad |
GB0207533D0 (en) | 2002-04-02 | 2002-05-08 | Oxford Glycosciences Uk Ltd | Protein |
US7745192B2 (en) | 2002-04-03 | 2010-06-29 | Venomics Pty Limited | Prothrombin activating protein |
EP1499352A4 (en) | 2002-04-12 | 2006-10-11 | Medimmune Inc | Recombinant anti-interleukin-9 antibodies |
EP2011886A3 (en) | 2002-04-16 | 2009-02-11 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
US20030206898A1 (en) | 2002-04-26 | 2003-11-06 | Steven Fischkoff | Use of anti-TNFalpha antibodies and another drug |
CA2486529A1 (en) | 2002-05-21 | 2003-11-27 | Dsm Ip Assets B.V. | Novel phospholipases and uses thereof |
JP4753578B2 (en) * | 2002-06-03 | 2011-08-24 | ジェネンテック, インコーポレイテッド | Synthetic antibody phage library |
EP2305710A3 (en) * | 2002-06-03 | 2013-05-29 | Genentech, Inc. | Synthetic antibody phage libraries |
EP2070949B1 (en) * | 2002-06-10 | 2013-01-16 | Vaccinex, Inc. | C35 antibodies and their use in the treatment of cancer |
EP1558086A4 (en) * | 2002-06-14 | 2008-03-05 | Univ Case Western Reserve | Cell targeting methods and compositions |
US7425618B2 (en) | 2002-06-14 | 2008-09-16 | Medimmune, Inc. | Stabilized anti-respiratory syncytial virus (RSV) antibody formulations |
WO2004001003A2 (en) | 2002-06-20 | 2003-12-31 | Board Of Trustees Operating Michigan State University | Plastid division and related genes and proteins, and methods of use |
ES2368733T3 (en) | 2002-07-18 | 2011-11-21 | Merus B.V. | RECOMBINANT PRODUCTION OF MIXTURES OF ANTIBODIES. |
USRE47770E1 (en) | 2002-07-18 | 2019-12-17 | Merus N.V. | Recombinant production of mixtures of antibodies |
KR20050042466A (en) | 2002-07-19 | 2005-05-09 | 애보트 바이오테크놀로지 리미티드 | Treatment of TNFα related disorders |
US7250551B2 (en) | 2002-07-24 | 2007-07-31 | President And Fellows Of Harvard College | Transgenic mice expressing inducible human p25 |
PL375301A1 (en) | 2002-08-10 | 2005-11-28 | Biogen Idec Ma Inc. | Nogo receptor antagonists |
US20040067532A1 (en) | 2002-08-12 | 2004-04-08 | Genetastix Corporation | High throughput generation and affinity maturation of humanized antibody |
US7425620B2 (en) | 2002-08-14 | 2008-09-16 | Scott Koenig | FcγRIIB-specific antibodies and methods of use thereof |
CN1675355A (en) | 2002-08-19 | 2005-09-28 | 帝斯曼知识产权资产管理有限公司 | Novel lipases and uses thereof |
JP2006514823A (en) | 2002-08-20 | 2006-05-18 | ミレニアム ファーマスーティカルズ インク | Compositions, kits and methods for identifying, evaluating, preventing and treating cervical cancer |
DE10238846A1 (en) * | 2002-08-20 | 2004-03-04 | Nemod Immuntherapie Ag | Active fusion proteins and processes for their production |
US7455989B2 (en) | 2002-08-20 | 2008-11-25 | Yeda Research And Development Co. Ltd. | AKAP84 and its use for visualization of biological structures |
WO2004019857A2 (en) * | 2002-08-29 | 2004-03-11 | Genentech, Inc. | Achaete-scute like-2 polypeptides and encoding nucleic acids and methods for the diagnosis and treatment of tumor |
AU2003295326A1 (en) | 2002-09-04 | 2004-04-23 | Cancer therapy using beta glucan and antibodies | |
US20050064508A1 (en) * | 2003-09-22 | 2005-03-24 | Semzyme | Peptide mediated synthesis of metallic and magnetic materials |
WO2005000194A2 (en) | 2002-10-08 | 2005-01-06 | Rinat Neuroscience Corp. | Methods for treating post-surgical pain by administering an anti-nerve growth factor antagonist antibody and compositions containing the same |
PT1556083E (en) | 2002-10-08 | 2011-03-17 | Rinat Neuroscience Corp | Methods for treating post-surgical pain by admisnistering an antibody against nerve growth factor and compositions containing the same |
WO2004032868A2 (en) * | 2002-10-09 | 2004-04-22 | Rinat Neuroscience Corp. | Methods of treating alzheimer's disease using antibodies directed against amyloid beta peptide and compositions thereof |
EP2891666B1 (en) | 2002-10-16 | 2017-06-28 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/O722P and methods of use thereof |
RU2377253C2 (en) * | 2002-12-02 | 2009-12-27 | Амген Фремонт,Инк. | Antibodies specific to tumour necrosis factor, and application thereof |
US7056702B2 (en) * | 2002-12-16 | 2006-06-06 | Kimberly Clark Co | Detecting lipocalin |
EP1581759B1 (en) * | 2002-12-23 | 2009-05-13 | Rinat Neuroscience Corp. | Methods for treating taxol-induced sensory neuropathy |
AU2003293958A1 (en) * | 2002-12-23 | 2004-07-22 | Apalexo Biotechnologie Gmbh | Purification of recombinant filamental bacteriophages by means of affinity chromatography to increase the immunogenicity and efficacy of phagic vaccines |
SI2263692T1 (en) | 2002-12-24 | 2020-10-30 | Rinat Neuroscience Corp. | Anti-NGF antibodies and methods using same |
US9498530B2 (en) | 2002-12-24 | 2016-11-22 | Rinat Neuroscience Corp. | Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same |
US7569364B2 (en) * | 2002-12-24 | 2009-08-04 | Pfizer Inc. | Anti-NGF antibodies and methods using same |
US20040209243A1 (en) | 2003-01-07 | 2004-10-21 | Andrew Nixon | Kunitz domain library |
AU2004205631A1 (en) * | 2003-01-16 | 2004-08-05 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2004065551A2 (en) * | 2003-01-21 | 2004-08-05 | Bristol-Myers Squibb Company | Polynucleotide encoding a novel acyl coenzyme a, monoacylglycerol acyltransferase-3 (mgat3), and uses thereof |
US7429472B2 (en) * | 2003-01-31 | 2008-09-30 | Promega Corporation | Method of immobilizing a protein or molecule via a mutant dehalogenase that is bound to an immobilized dehalogenase substrate and linked directly or indirectly to the protein or molecule |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
EP2369006B1 (en) | 2003-01-31 | 2014-03-12 | Promega Corporation | Covalent tethering of functional groups to proteins |
JP4764334B2 (en) * | 2003-02-01 | 2011-08-31 | タノックス インコーポレーテッド | High affinity anti-human IgE antibody |
DE602004030535D1 (en) | 2003-02-19 | 2011-01-27 | Rinat Neuroscience Corp | METHOD FOR TREATING PAIN BY ADMINISTERING A NERVE GROWTH FACTOR ANTAGONIST AND A NSAID AND COMPOSITION CONTAINING THEREOF |
DE602004027888D1 (en) | 2003-02-20 | 2010-08-12 | Seattle Genetics Inc | ANTI-CD70 ANTIBODY MEDICAMENT CONJUGATES AND THEIR |
US20040180387A1 (en) | 2003-03-13 | 2004-09-16 | Fujirebio Diagnostics, Inc. | Detection of urinary mesothelin-/megakaryocyte potentiating factor-related peptides for assessment of ovarian cancer |
NZ607886A (en) * | 2003-03-19 | 2014-09-26 | Biogen Idec Inc | Nogo receptor binding protein |
JP2006520806A (en) * | 2003-03-20 | 2006-09-14 | ライナット ニューロサイエンス コーポレイション | Methods for treating taxol-induced bowel disorder |
US7294701B2 (en) * | 2003-04-02 | 2007-11-13 | Technion Research & Development Foundation Ltd. | Antibody fragment capable of modulating multidrug resistance and compositions and kits and methods using same |
JP4764818B2 (en) | 2003-04-11 | 2011-09-07 | メディミューン,エルエルシー | Recombinant IL-9 antibody and use thereof |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
US20050014932A1 (en) | 2003-05-15 | 2005-01-20 | Iogenetics, Llc | Targeted biocides |
US20100069614A1 (en) | 2008-06-27 | 2010-03-18 | Merus B.V. | Antibody producing non-human mammals |
KR20060006975A (en) | 2003-05-30 | 2006-01-20 | 제넨테크, 인크. | Treatment with anti-vegf antibodies |
CA2527694C (en) | 2003-05-30 | 2015-07-14 | Hendricus Renerus Jacobus Mattheus Hoogenboom | Fab library for the preparation of anti vegf and anti rabies virus fabs |
US9708410B2 (en) | 2003-05-30 | 2017-07-18 | Janssen Biotech, Inc. | Anti-tissue factor antibodies and compositions |
ES2323425T3 (en) | 2003-06-06 | 2009-07-15 | Genentech, Inc. | MODULATION OF THE INTERACTION BETWEEN THE BETA CHAIN OF HGF AND C-MET. |
CA2542232A1 (en) | 2003-06-09 | 2005-01-20 | Alnylam Pharmaceuticals, Inc. | Method for treating neurodegenerative disease by inhibiting alpha-synuclein |
WO2005005601A2 (en) * | 2003-06-09 | 2005-01-20 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
NZ544317A (en) | 2003-07-08 | 2009-05-31 | Genentech Inc | IL-17 A/F heterologous polypeptides and therapeutic uses thereof |
AU2004259727A1 (en) * | 2003-07-15 | 2005-02-03 | Barros Research Institute | Compositions and methods for immunotherapy of cancer and infectious diseases. |
WO2005010163A2 (en) * | 2003-07-15 | 2005-02-03 | Barros Research Institute | Compositions and methods for immunotherapy of human immunotherapy of human immunodeficiency virus (hiv) |
US7727752B2 (en) | 2003-07-29 | 2010-06-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US20050106667A1 (en) | 2003-08-01 | 2005-05-19 | Genentech, Inc | Binding polypeptides with restricted diversity sequences |
US7758859B2 (en) | 2003-08-01 | 2010-07-20 | Genentech, Inc. | Anti-VEGF antibodies |
HN2004000285A (en) | 2003-08-04 | 2006-04-27 | Pfizer Prod Inc | ANTIBODIES DIRECTED TO c-MET |
US20050221383A1 (en) | 2003-08-08 | 2005-10-06 | Choong-Chin Liew | Osteoarthritis biomarkers and uses thereof |
EP2272566A3 (en) | 2003-08-18 | 2013-01-02 | MedImmune, LLC | Humanisation of antibodies |
WO2005023831A1 (en) * | 2003-09-05 | 2005-03-17 | The Scripps Research Institute | Detection of cholesterol ozonation products |
CA2537976A1 (en) * | 2003-09-05 | 2005-03-17 | The Scripps Research Institute | Ozonation products of cholesterol for the treatment and prevention of artherosclerosis and/or cardiovascular diseases |
AR045563A1 (en) | 2003-09-10 | 2005-11-02 | Warner Lambert Co | ANTIBODIES DIRECTED TO M-CSF |
IL158287A0 (en) | 2003-10-07 | 2004-05-12 | Yeda Res & Dev | Antibodies to nik, their preparation and use |
EP2051077A3 (en) | 2003-10-07 | 2009-05-27 | Millennium Pharmaceuticals, Inc. | Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer |
EP2441474B1 (en) | 2003-10-17 | 2015-08-05 | Joslin Diabetes Center, Inc. | Methods and compositions for modulating adipocyte function |
US7329725B1 (en) * | 2003-10-29 | 2008-02-12 | Nastech Pharmaceutical Company Inc. | Phage displayed Trp cage ligands |
GB0325836D0 (en) * | 2003-11-05 | 2003-12-10 | Celltech R&D Ltd | Biological products |
US20050100965A1 (en) | 2003-11-12 | 2005-05-12 | Tariq Ghayur | IL-18 binding proteins |
CA2545603A1 (en) | 2003-11-12 | 2005-05-26 | Biogen Idec Ma Inc. | Neonatal fc receptor (fcrn)-binding polypeptide variants, dimeric fc binding proteins and methods related thereto |
ES2472690T3 (en) | 2003-11-17 | 2014-07-02 | Genentech, Inc. | Antibody against CD22 for the treatment of tumors of hematopoietic origin |
EP1687026B1 (en) | 2003-11-21 | 2008-05-14 | UCB Pharma, S.A. | Method for the treatment of multiple sclerosis by inhibiting il-17 activity |
JP5042631B2 (en) * | 2003-12-04 | 2012-10-03 | バクシネックス インコーポレーティッド | Method of killing tumor cells by targeting intracellular antigens exposed on apoptotic tumor cells |
EP2336178A1 (en) | 2003-12-11 | 2011-06-22 | Genentech, Inc. | Methods and compositions for inhibiting C-Met dimerization and activation |
KR20070001932A (en) * | 2003-12-23 | 2007-01-04 | 리나트 뉴로사이언스 코퍼레이션 | Agonist anti-trkc antibodies and methods using same |
GB0329825D0 (en) * | 2003-12-23 | 2004-01-28 | Celltech R&D Ltd | Biological products |
US20050266425A1 (en) * | 2003-12-31 | 2005-12-01 | Vaccinex, Inc. | Methods for producing and identifying multispecific antibodies |
DK2177537T3 (en) | 2004-01-09 | 2011-12-12 | Pfizer | Antibodies to MAdCAM |
MXPA06008126A (en) | 2004-01-14 | 2008-02-14 | Univ Ohio | Methods of producing peptides/proteins in plants and peptides/proteins produced thereby. |
EP1761561B1 (en) | 2004-01-20 | 2015-08-26 | KaloBios Pharmaceuticals, Inc. | Antibody specificity transfer using minimal essential binding determinants |
CA2554054C (en) | 2004-01-20 | 2013-06-04 | Merus B.V. | Mixtures of binding proteins |
DK2842576T3 (en) | 2004-01-21 | 2017-10-16 | Novo Nordisk Healthcare Ag | Transglutaminase-mediated peptide conjugation |
US20060014211A1 (en) | 2004-01-21 | 2006-01-19 | Fujirebio Diagnostics, Inc. | Detection of mesothelin-/megakaryocyte potentiating factor-related peptides for assessment of the peritoneum and the peritoneal cavity |
MXPA06008496A (en) | 2004-02-02 | 2007-01-30 | Ambrx Inc | Modified human interferon polypeptides and their uses. |
WO2005074633A2 (en) * | 2004-02-03 | 2005-08-18 | The Regents Of The University Of Michigan | Compositions and methods for characterizing, regulating, diagnosing, and treating cancer |
PL1729795T3 (en) | 2004-02-09 | 2016-08-31 | Human Genome Sciences Inc | Albumin fusion proteins |
EP2168986A3 (en) | 2004-02-19 | 2010-07-28 | Genentech, Inc. | CDR-repaired antibodies |
WO2005085288A2 (en) | 2004-03-01 | 2005-09-15 | The Cbr Institute For Biomedical Research | Natural igm antibodies and inhibitors thereof |
EP1735453A2 (en) * | 2004-03-12 | 2006-12-27 | The Scripps Research Institute | Fluorescent signal emitting live cell biosensor molecules and dyes for detection and quantification of protein activities |
MXPA06011020A (en) | 2004-03-24 | 2007-08-14 | Tripath Imaging Inc | Methods and compositions for the detection of cervical disease. |
US7973139B2 (en) * | 2004-03-26 | 2011-07-05 | Human Genome Sciences, Inc. | Antibodies against nogo receptor |
CN1961003B (en) * | 2004-03-31 | 2013-03-27 | 健泰科生物技术公司 | Humanized anti-TGF-beta antibodies |
US7794713B2 (en) | 2004-04-07 | 2010-09-14 | Lpath, Inc. | Compositions and methods for the treatment and prevention of hyperproliferative diseases |
NZ549990A (en) * | 2004-04-07 | 2009-08-28 | Rinat Neuroscience Copr | Methods for treating bone cancer pain by administering a nerve growth factor antagonist |
US7785903B2 (en) * | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
TW201705980A (en) | 2004-04-09 | 2017-02-16 | 艾伯維生物技術有限責任公司 | Multiple-variable dose regimen for treating TNF[alpha]-related disorders |
EP1745149A4 (en) | 2004-04-15 | 2008-08-06 | Univ Florida | Neural proteins as biomarkers for nervous system injury and other neural disorders |
US20060003348A1 (en) * | 2004-04-16 | 2006-01-05 | Genentech. Inc. | Omi PDZ modulators |
EP1751177A4 (en) | 2004-04-19 | 2008-07-16 | Univ Ohio | Cross-linkable glycoproteins and methods of making the same |
US8318905B2 (en) | 2004-04-23 | 2012-11-27 | Richard Kroczek | Antibodies for depletion of ICOS-positive cells in vivo |
EP1789453A2 (en) * | 2004-05-18 | 2007-05-30 | Genentech, Inc. | M13 virus major coat protein variants for c-terminal and bi-terminal display of a heterologous protein |
EP1602928A1 (en) * | 2004-06-01 | 2005-12-07 | Universiteit Maastricht | Process and kit for determining binding parameters of bioaffinity binding reactions |
AU2005250511B2 (en) * | 2004-06-03 | 2012-01-19 | Phylogica Limited | Peptide modulators of cellular phenotype and bi-nucleic acid fragment library |
US20090081243A1 (en) | 2004-06-03 | 2009-03-26 | Athlomics Pty Ltd. | Agents and methods for diagnosing stress |
US7604947B2 (en) * | 2004-06-09 | 2009-10-20 | Cornell Research Foundation, Inc. | Detection and modulation of cancer stem cells |
MXPA06014684A (en) * | 2004-06-18 | 2007-02-12 | Ambrx Inc | Novel antigen-binding polypeptides and their uses. |
GB0414054D0 (en) | 2004-06-23 | 2004-07-28 | Owen Mumford Ltd | Improvements relating to automatic injection devices |
BRPI0512500A (en) | 2004-06-24 | 2008-03-11 | Biogen Idec Inc | treatment or conditions involving demyelination |
US6986264B1 (en) * | 2004-07-15 | 2006-01-17 | Carrier Corporation | Economized dehumidification system |
EP2322215A3 (en) | 2004-07-16 | 2011-09-28 | Pfizer Products Inc. | Combination treatment for non-hematologic malignancies using an anti-IGF-1R antibody |
ATE456579T1 (en) * | 2004-07-20 | 2010-02-15 | Genentech Inc | ANGIOPOIETIN-LIKE 4 PROTEIN INHIBITOR COMBINATIONS AND THEIR USE |
US8604185B2 (en) | 2004-07-20 | 2013-12-10 | Genentech, Inc. | Inhibitors of angiopoietin-like 4 protein, combinations, and their use |
WO2009100255A2 (en) | 2008-02-08 | 2009-08-13 | Ambrx, Inc. | Modified leptin polypeptides and their uses |
KR20070039593A (en) * | 2004-07-21 | 2007-04-12 | 암브룩스, 인코포레이티드 | Biosynthetic polypeptides utilizing non-naturally encoded amino acids |
US7342093B2 (en) | 2004-07-23 | 2008-03-11 | University Of Massachusetts | Compounds that inhibit Hsp90 protein-protein interactions with IAP proteins |
US7425436B2 (en) | 2004-07-30 | 2008-09-16 | Promega Corporation | Covalent tethering of functional groups to proteins and substrates therefor |
US20070087400A1 (en) * | 2004-07-30 | 2007-04-19 | Aldis Darzins | Covalent tethering of functional groups to proteins and substrates therefor |
SG190665A1 (en) * | 2004-07-30 | 2013-06-28 | Rinat Neuroscience Corp | Antibodies directed against amyloid-beta peptide and methods using same |
JP5060293B2 (en) | 2004-08-03 | 2012-10-31 | バイオジェン・アイデック・エムエイ・インコーポレイテッド | TAJ in neural function |
SI1784426T1 (en) | 2004-09-03 | 2012-03-30 | Genentech Inc | Humanized anti-beta7 antagonists and uses therefor |
US7700720B2 (en) | 2004-09-21 | 2010-04-20 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
WO2006047417A2 (en) | 2004-10-21 | 2006-05-04 | University Of Florida Research Foundation, Inc. | Detection of cannabinoid receptor biomarkers and uses thereof |
NZ596663A (en) | 2004-10-21 | 2013-07-26 | Genentech Inc | Use of vegf antagonists in intraocular neovascular disease treatment |
CA2585717A1 (en) | 2004-10-27 | 2006-05-04 | Medimmune Inc. | Modulation of antibody specificity by tailoring the affinity to cognate antigens |
GB0426146D0 (en) | 2004-11-29 | 2004-12-29 | Bioxell Spa | Therapeutic peptides and method |
WO2006066088A2 (en) * | 2004-12-16 | 2006-06-22 | Genentech, Inc. | Methods for treating autoimmune disorders |
MX2007007580A (en) * | 2004-12-22 | 2007-12-11 | Ambrx Inc | Modified human growth hormone. |
BRPI0518661A2 (en) * | 2004-12-22 | 2008-12-02 | Ambrx Inc | Methods for expression and purification of recombinant human growth hormone |
KR101224781B1 (en) | 2004-12-22 | 2013-01-21 | 암브룩스, 인코포레이티드 | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
ATE541934T1 (en) * | 2004-12-22 | 2012-02-15 | Ambrx Inc | AMINOACYL TRNA SYNTHETASE COMPOSITIONS AND USES THEREOF |
EP1833970A2 (en) | 2004-12-22 | 2007-09-19 | Genentech, Inc. | Methods for producing soluble multi-membrane-spanning proteins |
EP1752471B9 (en) | 2005-01-05 | 2009-04-15 | f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. | Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions |
EP1838736B1 (en) | 2005-01-05 | 2013-03-06 | Biogen Idec MA Inc. | Cripto binding molecules |
GT200600031A (en) | 2005-01-28 | 2006-08-29 | ANTI-BETA ANTIBODY FORMULATION | |
US8029783B2 (en) * | 2005-02-02 | 2011-10-04 | Genentech, Inc. | DR5 antibodies and articles of manufacture containing same |
CA2599589A1 (en) | 2005-02-07 | 2006-08-17 | Genenews,Inc. | Mild osteoarthritis biomarkers and uses thereof |
DK1853718T3 (en) | 2005-02-15 | 2015-11-09 | Univ Duke | ANTI-CD19 ANTIBODIES AND THEIR USE IN ONCOLOGY |
JP2008531730A (en) * | 2005-03-04 | 2008-08-14 | キュアーディーエム、インク. | Methods and pharmaceutical compositions for treating type I diabetes mellitus and other conditions |
US20090142338A1 (en) * | 2005-03-04 | 2009-06-04 | Curedm, Inc. | Methods and Compositions for Treating Type 1 and Type 2 Diabetes Mellitus and Related Conditions |
RU2007137489A (en) | 2005-03-10 | 2009-04-20 | Дженентек, Инк. (Us) | METHODS AND COMPOSITIONS FOR MODULATION OF VESSEL INTEGRITY |
EP1869192B1 (en) | 2005-03-18 | 2016-01-20 | MedImmune, LLC | Framework-shuffling of antibodies |
WO2006102557A2 (en) | 2005-03-22 | 2006-09-28 | The President And Fellows Of Harvard College | Treatment of protein degradation disorders |
EP2535355B1 (en) | 2005-03-23 | 2019-01-02 | Genmab A/S | Antibodies against CD38 for treatment of multiple myeloma |
GB0506912D0 (en) | 2005-04-05 | 2005-05-11 | Celltech R&D Ltd | Biological products |
WO2006113665A2 (en) | 2005-04-15 | 2006-10-26 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
US20060269556A1 (en) * | 2005-04-18 | 2006-11-30 | Karl Nocka | Mast cell activation using siglec 6 antibodies |
CA2605507C (en) | 2005-04-19 | 2016-06-28 | Seattle Genetics, Inc. | Humanized anti-cd70 binding agents and uses thereof |
US7807159B2 (en) | 2005-04-25 | 2010-10-05 | Amgen Fremont Inc. | Antibodies to myostatin |
AU2006238930B2 (en) | 2005-04-26 | 2010-12-23 | Pfizer Inc. | P-cadherin antibodies |
AR054260A1 (en) * | 2005-04-26 | 2007-06-13 | Rinat Neuroscience Corp | METHODS OF TREATMENT OF DISEASES OF THE LOWER MOTOR NEURONE AND COMPOSITIONS USED IN THE SAME |
US7595380B2 (en) | 2005-04-27 | 2009-09-29 | Tripath Imaging, Inc. | Monoclonal antibodies and methods for their use in the detection of cervical disease |
UY29504A1 (en) | 2005-04-29 | 2006-10-31 | Rinat Neuroscience Corp | DIRECTED ANTIBODIES AGAINST BETA AMYLOID PEPTIDE AND METHODS USING THE SAME. |
JO3058B1 (en) | 2005-04-29 | 2017-03-15 | Applied Molecular Evolution Inc | Anti-IL-6 Antibodies,Compositions,Methods and uses |
CN101500607B (en) | 2005-05-16 | 2013-11-27 | 阿布维生物技术有限公司 | Use of TNFalpha inhibitor for treatment of erosive polyarthritis |
NZ581779A (en) | 2005-05-17 | 2011-09-30 | Univ Connecticut | Composition and methods for immunomodulation in an organism comprising interleukin-15 polypeptide and interleukin-15 receptor subunit A polypeptide complex |
EP2295066B1 (en) | 2005-05-25 | 2016-04-27 | CureDM Group Holdings, LLC | Peptides, derivatives and analogs thereof, and methods of using same |
DK1888113T3 (en) | 2005-05-27 | 2014-09-01 | Biogen Idec Inc | TWEAK BINDING ANTIBODIES |
EP1893229B1 (en) * | 2005-06-03 | 2011-10-19 | Ambrx, Inc. | Improved human interferon molecules and their uses |
NZ563341A (en) | 2005-06-06 | 2009-10-30 | Genentech Inc | Methods for identifying agents that modulate a gene that encodes for a PRO1568 polypeptide |
WO2006135886A2 (en) * | 2005-06-13 | 2006-12-21 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
CN101365722A (en) | 2005-06-17 | 2009-02-11 | 艾兰制药国际有限公司 | Methods of purifying anti a beta antibodies |
AU2006261920A1 (en) | 2005-06-23 | 2007-01-04 | Medimmune, Llc | Antibody formulations having optimized aggregation and fragmentation profiles |
WO2007005955A2 (en) | 2005-06-30 | 2007-01-11 | Centocor, Inc. | Anti-il-23 antibodies, compositions, methods and uses |
CN103145842A (en) | 2005-06-30 | 2013-06-12 | Abbvie公司 | Il-12/p40 binding proteins |
NZ564923A (en) | 2005-07-07 | 2012-02-24 | Athlomics Pty Ltd | Polynucleotide marker genes and their expression, for diagnosis of endotoxemia |
EP1904652A2 (en) * | 2005-07-08 | 2008-04-02 | Brystol-Myers Squibb Company | Single nucleotide polymorphisms associated with dose-dependent edema and methods of use thereof |
PT1904104E (en) | 2005-07-08 | 2013-11-21 | Biogen Idec Inc | Sp35 antibodies and uses thereof |
CA2615615A1 (en) | 2005-07-22 | 2007-02-01 | Y's Therapeutics Co., Ltd. | Anti-cd26 antibodies and methods of use thereof |
ATE470151T1 (en) | 2005-08-02 | 2010-06-15 | Xbiotech Inc | DIAGNOSIS, TREATMENT AND PREVENTION OF VASCULAR DISEASE USING IL-1ALPHA AUTOANTIBODIES |
NZ565631A (en) | 2005-08-03 | 2011-01-28 | Adelaide Res & Innovation Pty | Polysaccharide synthases |
JP2009504183A (en) | 2005-08-15 | 2009-02-05 | ジェネンテック・インコーポレーテッド | Gene disruption and related compositions and methods |
AU2005335491B2 (en) | 2005-08-18 | 2010-11-25 | Ambrx, Inc. | Compositions of tRNA and uses thereof |
BRPI0615026A8 (en) | 2005-08-19 | 2018-03-06 | Abbott Lab | double variable domain immunoglobulin and its uses |
US20070041905A1 (en) | 2005-08-19 | 2007-02-22 | Hoffman Rebecca S | Method of treating depression using a TNF-alpha antibody |
US20070202512A1 (en) * | 2005-08-19 | 2007-08-30 | Bristol-Myers Squibb Company | Human single nucleotide polymorphisms associated with dose-dependent weight gain and methods of use thereof |
EP2500358A3 (en) | 2005-08-19 | 2012-10-17 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
US20090215992A1 (en) * | 2005-08-19 | 2009-08-27 | Chengbin Wu | Dual variable domain immunoglobulin and uses thereof |
US7612181B2 (en) * | 2005-08-19 | 2009-11-03 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
PT2447283E (en) | 2005-09-07 | 2015-10-08 | Pfizer | Human monoclonal antibodies to activin receptor-like kinase-1 (alk-1) |
WO2007031734A1 (en) * | 2005-09-14 | 2007-03-22 | Ucb Pharma S.A. | Comb polymers |
EP1928905B1 (en) | 2005-09-30 | 2015-04-15 | AbbVie Deutschland GmbH & Co KG | Binding domains of proteins of the repulsive guidance molecule (rgm) protein family and functional fragments thereof, and their use |
KR101176830B1 (en) | 2005-10-17 | 2012-08-27 | 주식회사 아이지세라피 | Novel method for phage display |
TW200730825A (en) | 2005-10-21 | 2007-08-16 | Genenews Inc | Method and apparatus for correlating levels of biomarker products with disease |
ES2526705T3 (en) | 2005-10-25 | 2015-01-14 | The Johns Hopkins University | Methods and compositions for the treatment of Marfan syndrome and associated disorders |
PT1978993T (en) | 2005-10-31 | 2017-03-17 | Oncomed Pharm Inc | Compositions and methods for treating cancer based on human fzd receptors |
US7723477B2 (en) | 2005-10-31 | 2010-05-25 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for inhibiting Wnt-dependent solid tumor cell growth |
US7723112B2 (en) * | 2005-10-31 | 2010-05-25 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing cancer |
WO2007089303A2 (en) | 2005-11-01 | 2007-08-09 | Abbott Biotechnology Ltd. | Methods and compositions for diagnosing ankylosing spondylitis using biomarkers |
KR20080080109A (en) | 2005-11-04 | 2008-09-02 | 바이오겐 아이덱 엠에이 인코포레이티드 | Methods for promoting neurite outgrowth and survival of dopaminergic neurons |
EA014900B1 (en) | 2005-11-07 | 2011-02-28 | Зе Скрипс Ресеч Инститьют | Compositions and methods for controlling tissue factor signaling specificity |
AU2006311568B2 (en) * | 2005-11-08 | 2010-11-11 | Ambrx, Inc. | Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides |
UA96139C2 (en) | 2005-11-08 | 2011-10-10 | Дженентек, Інк. | Anti-neuropilin-1 (nrp1) antibody |
EP3069731A1 (en) | 2005-11-14 | 2016-09-21 | Labrys Biologics Inc. | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
US7807790B2 (en) | 2005-11-14 | 2010-10-05 | Metamol Theranostics, Llc | Peptide sequence that promotes tumor invasion |
PT2339014E (en) * | 2005-11-16 | 2015-10-13 | Ambrx Inc | Methods and compositions comprising non-natural amino acids |
WO2007081608A2 (en) | 2005-11-21 | 2007-07-19 | Genentech, Inc. | Novel gene disruptions, compositions and methods relating thereto |
JP5322653B2 (en) | 2005-11-28 | 2013-10-23 | ザイモジェネティクス, インコーポレイテッド | IL-21 antagonist |
JP5352237B2 (en) | 2005-11-30 | 2013-11-27 | マサチューセッツ インスティテュート オブ テクノロジー | Pathogen detection biosensor |
KR20140087058A (en) | 2005-11-30 | 2014-07-08 | 애브비 인코포레이티드 | Monoclonal antibodies against amyloid beta protein and uses thereof |
PT2289909E (en) | 2005-11-30 | 2015-02-10 | Abbvie Inc | Screening method, process for purifying of non-diffusible a-beta oligomers, selective antibodies against said non-diffusible a-beta oligomers and a process for manufacturing of said antibodies |
EP1965827B1 (en) | 2005-12-02 | 2015-02-25 | Biogen Idec MA Inc. | Treatment of conditions involving demyelination |
CA2631327C (en) * | 2005-12-02 | 2015-10-13 | Genentech, Inc. | Her2 binding polypeptides and uses thereof |
WO2007126439A2 (en) | 2005-12-02 | 2007-11-08 | Genentech, Inc. | Compositions and methods for the treatment of diseases and disorders associated with cytokine signaling involving antibodies that bind to il-22 and il-22r |
PT1960430E (en) | 2005-12-09 | 2015-01-05 | Ucb Pharma Sa | Antibody molecules having specificity for human il-6 |
EP1968635B1 (en) * | 2005-12-14 | 2014-09-17 | Ambrx, Inc. | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
US20070264687A1 (en) | 2005-12-15 | 2007-11-15 | Min-Yuan Chou | Recombinant triplex scaffold-based polypeptides |
US10183986B2 (en) | 2005-12-15 | 2019-01-22 | Industrial Technology Research Institute | Trimeric collagen scaffold antibodies |
EP1973948B1 (en) | 2005-12-15 | 2015-02-11 | Genentech, Inc. | Methods and compositions for targeting polyubiquitin |
US7632498B2 (en) | 2005-12-19 | 2009-12-15 | Tripath Imaging, Inc. | MCM6 and MCM7 monoclonal antibodies and methods for their use in the detection of cervical disease |
EA035459B1 (en) | 2005-12-29 | 2020-06-19 | Сентокор, Инк. | ANTI-IL-23p19 ANTIBODY |
SI1973950T1 (en) | 2006-01-05 | 2015-01-30 | Genentech, Inc. | Anti-ephb4 antibodies and methods using the same |
WO2007084321A2 (en) | 2006-01-12 | 2007-07-26 | Alexion Pharmaceuticals, Inc. | Antibodies to ox-2/cd200 and uses thereof |
WO2007089601A2 (en) | 2006-01-27 | 2007-08-09 | Biogen Idec Ma Inc. | Nogo receptor antagonists |
CA2637446A1 (en) | 2006-01-27 | 2007-08-09 | Tripath Imaging, Inc. | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
WO2007090126A2 (en) * | 2006-01-30 | 2007-08-09 | Invitrogen Corporation | Compositions and methods for detecting and quantifying toxic substances in disease states |
US20070175313A1 (en) * | 2006-01-31 | 2007-08-02 | Kevin Vandervliet | MP3 player holder assembly |
KR20090039666A (en) | 2006-02-01 | 2009-04-22 | 아라나 테라퓨틱스 리미티드 | Domain antibody construct |
CN101400362B (en) | 2006-02-14 | 2016-10-12 | 哈佛大学校长及研究员协会 | Bifunctional histone deacetylase inhibitors |
US20080311107A1 (en) | 2006-02-17 | 2008-12-18 | Genetech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
CA2950465A1 (en) * | 2006-02-20 | 2007-08-30 | Phylogica Limited | Method of constructing and screening libraries of peptide structures |
US20080019961A1 (en) * | 2006-02-21 | 2008-01-24 | Regents Of The University Of Michigan | Hedgehog signaling pathway antagonist cancer treatment |
WO2007100640A2 (en) * | 2006-02-21 | 2007-09-07 | The Regents Of The University Of Michigan | Growth hormone receptor antagonist cancer treatment |
TW200744634A (en) | 2006-02-21 | 2007-12-16 | Wyeth Corp | Methods of using antibodies against human IL-22 |
TWI417301B (en) | 2006-02-21 | 2013-12-01 | Wyeth Corp | Antibodies against human il-22 and uses therefor |
WO2007100711A2 (en) * | 2006-02-24 | 2007-09-07 | Investigen, Inc. | Methods and compositions for detecting polynucleotides |
EP2650306A1 (en) | 2006-03-06 | 2013-10-16 | Aeres Biomedical Limited | Humanized Anti-CD22 antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
AR059851A1 (en) | 2006-03-16 | 2008-04-30 | Genentech Inc | ANTIBODIES OF EGFL7 AND METHODS OF USE |
EP2010569A4 (en) | 2006-03-20 | 2009-09-09 | Xoma Technology Ltd | Human antibodies specific for gastrin materials and methods |
WO2007126455A2 (en) | 2006-04-05 | 2007-11-08 | Genentech, Inc. | Method for using boc/cdo to modulate hedgehog signaling |
NZ611859A (en) | 2006-04-05 | 2014-12-24 | Abbvie Biotechnology Ltd | Antibody purification |
WO2008063213A2 (en) | 2006-04-10 | 2008-05-29 | Abbott Biotechnology Ltd. | Uses and compositions for treatment of psoriatic arthritis |
US7695928B2 (en) | 2006-04-10 | 2010-04-13 | Genentech, Inc. | Disheveled PDZ modulators |
EP2708242A3 (en) | 2006-04-10 | 2014-03-26 | Abbott Biotechnology Ltd | Uses and compositions for treatment of ankylosing spondylitis |
CA2564435A1 (en) | 2006-04-10 | 2007-10-10 | Abbott Biotechnology Ltd. | Methods for monitoring and treating intestinal disorders |
US20090288176A1 (en) | 2006-04-19 | 2009-11-19 | Genentech, Inc. | Novel Gene Disruptions, Compositions and Methods Relating Thereto |
US10522240B2 (en) | 2006-05-03 | 2019-12-31 | Population Bio, Inc. | Evaluating genetic disorders |
US7702468B2 (en) | 2006-05-03 | 2010-04-20 | Population Diagnostics, Inc. | Evaluating genetic disorders |
JP5497431B2 (en) | 2006-05-03 | 2014-05-21 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Histone deacetylase and tubulin deacetylase inhibitors |
JP2009536527A (en) * | 2006-05-09 | 2009-10-15 | ジェネンテック・インコーポレーテッド | Binding polypeptide with optimized scaffold |
WO2007134132A2 (en) * | 2006-05-12 | 2007-11-22 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of bladder and urinary tract tumors |
MX2008015132A (en) | 2006-05-30 | 2008-12-10 | Genentech Inc | Antibodies and immunoconjugates and uses therefor. |
US7862812B2 (en) | 2006-05-31 | 2011-01-04 | Lpath, Inc. | Methods for decreasing immune response and treating immune conditions |
GB0611116D0 (en) | 2006-06-06 | 2006-07-19 | Oxford Genome Sciences Uk Ltd | Proteins |
MX2008015541A (en) | 2006-06-06 | 2008-12-18 | Genentech Inc | Anti-dll4 antibodies and methods using same. |
WO2007146172A2 (en) | 2006-06-07 | 2007-12-21 | Bioalliance C.V. | Antibodies recognizing a carbohydrate containing epitope on cd-43 and cea expressed on cancer cells and methods using same |
BRPI0713426A2 (en) | 2006-06-14 | 2012-10-09 | Macrogenics Inc | methods of treating, slowing the progression, or ameliorating one or more symptoms of a disorder, and preventing or delaying the onset of a disorder |
EP2029173B1 (en) | 2006-06-26 | 2016-07-20 | MacroGenics, Inc. | Fc riib-specific antibodies and methods of use thereof |
DE102006030028A1 (en) * | 2006-06-29 | 2008-02-14 | Forschungszentrum Jülich GmbH | A method for finding bait-binding specific molecules and bait-binding molecules and their use |
BRPI0713802A2 (en) | 2006-06-30 | 2012-11-06 | Abbott Biotech Ltd | automatic injection device |
US7572618B2 (en) | 2006-06-30 | 2009-08-11 | Bristol-Myers Squibb Company | Polynucleotides encoding novel PCSK9 variants |
DK2046951T5 (en) | 2006-07-05 | 2012-01-23 | Catalyst Biosciences Inc | Protease screening methods and proteases identified thereby |
AT503889B1 (en) | 2006-07-05 | 2011-12-15 | Star Biotechnologische Forschungs Und Entwicklungsges M B H F | MULTIVALENT IMMUNE LOBULINE |
AU2007274738B2 (en) | 2006-07-18 | 2013-11-28 | Sanofi-Aventis | Antagonist antibody against EphA2 for the treatment of cancer |
PL2511301T3 (en) | 2006-08-04 | 2018-05-30 | Medimmune Limited | Human antibodies to erbb 2 |
EP2520935A3 (en) | 2006-08-09 | 2013-02-13 | Homestead Clinical Corporation | Organ-specific proteins and methods of their use |
CA2661782C (en) | 2006-08-28 | 2019-04-16 | La Jolla Institute For Allergy And Immunology | Antagonistic human light-specific human monoclonal antibodies |
CA2662629C (en) | 2006-09-07 | 2019-08-13 | University Of Otago | Bnp signal peptide fragments for acute cardiac disorders |
CN106008699A (en) | 2006-09-08 | 2016-10-12 | Ambrx公司 | Modified human plasma polypeptide or Fc scaffolds and their uses |
CA2662753C (en) * | 2006-09-08 | 2016-02-23 | Ambrx, Inc. | Hybrid suppressor trna for vertebrate cells |
US8420792B2 (en) * | 2006-09-08 | 2013-04-16 | Ambrx, Inc. | Suppressor tRNA transcription in vertebrate cells |
UA115964C2 (en) | 2006-09-08 | 2018-01-25 | Еббві Айрленд Анлімітед Компані | INTERLAYKIN-13-Binding Protein |
US20100008910A1 (en) | 2006-09-12 | 2010-01-14 | John Chant | Methods and compositions for the diagnosis and treatment of cancer |
WO2008034161A1 (en) * | 2006-09-19 | 2008-03-27 | Phylogica Limited | Neuroprotective peptide inhibitors of ap-1 signaling and uses therefor |
NZ576122A (en) | 2006-09-26 | 2012-09-28 | Genmab As | Anti-cd38 plus corticosteroids plus a non-corticosteroid chemotherapeutic for treating tumors |
WO2008042236A2 (en) | 2006-09-29 | 2008-04-10 | Oncomed Pharmaceuticals, Inc. | Compositions and methods for diagnosing and treating cancer |
CN101522219A (en) | 2006-10-12 | 2009-09-02 | 惠氏公司 | Methods and compositions with reduced opalescence |
NZ576032A (en) | 2006-10-12 | 2012-03-30 | Genentech Inc | Antibodies to lymphotoxin-alpha |
EP2407548A1 (en) | 2006-10-16 | 2012-01-18 | MedImmune, LLC | Molecules with reduced half-lives, compositions and uses thereof |
GB0620729D0 (en) | 2006-10-18 | 2006-11-29 | Ucb Sa | Biological products |
EP1914242A1 (en) * | 2006-10-19 | 2008-04-23 | Sanofi-Aventis | Novel anti-CD38 antibodies for the treatment of cancer |
US20080096193A1 (en) * | 2006-10-24 | 2008-04-24 | Charles Robert Bupp | Methods and compositions for detecting polynucleotides |
ES2636089T3 (en) | 2006-10-27 | 2017-10-05 | Genentech, Inc. | Antibodies and immunoconjugates and uses for them |
US8420367B2 (en) | 2006-10-30 | 2013-04-16 | Promega Corporation | Polynucleotides encoding mutant hydrolase proteins with enhanced kinetics and functional expression |
EP2064315B1 (en) | 2006-11-03 | 2015-05-13 | Wyeth LLC | Glycolysis-inhibiting substances in cell culture |
EP2094282A4 (en) | 2006-11-15 | 2010-05-05 | Functional Genetics Inc | Anti-tsg101 antibodies and their uses for treatment of viral infections |
WO2008064306A2 (en) * | 2006-11-22 | 2008-05-29 | Curedm, Inc. | Methods and compositions relating to islet cell neogenesis |
US7488807B2 (en) | 2006-11-22 | 2009-02-10 | 3M Innovative Properties Company | Antibody with protein A selectivity |
US8067179B2 (en) * | 2006-11-30 | 2011-11-29 | Research Development Foundation | Immunoglobulin libraries |
US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
EP2609932B1 (en) | 2006-12-01 | 2022-02-02 | Seagen Inc. | Variant target binding agents and uses thereof |
EP2121979B1 (en) | 2006-12-05 | 2017-07-12 | Decode Genetics EHF | Genetic markers for risk management of cardiac arrhythmia |
EP2687232A1 (en) | 2006-12-06 | 2014-01-22 | MedImmune, LLC | Methods of treating systemic lupus erythematosus |
MY183404A (en) | 2006-12-19 | 2021-02-18 | Genentech Inc | Vegf-specific antagonists for adjuvant and neoadjuvant therapy and the treatment of early stage tumors |
EP2094306A2 (en) | 2006-12-20 | 2009-09-02 | XOMA Technology Ltd. | Treatment of il-1-beta related diseases |
WO2008079849A2 (en) * | 2006-12-22 | 2008-07-03 | Genentech, Inc. | Antibodies to insulin-like growth factor receptor |
EP2913341A1 (en) | 2006-12-22 | 2015-09-02 | University of Utah Research Foundation | Method of detecting ocular diseases and pathologic conditions and treatment of same |
JP5623747B2 (en) | 2006-12-27 | 2014-11-12 | エモリー ユニバーシティ | Compositions and methods for treating infections and tumors |
EP2114443A4 (en) | 2006-12-29 | 2011-08-10 | Abbott Lab | Dual-specific il-1a/ il-1b antibodies |
LT2740744T (en) | 2007-01-09 | 2018-05-10 | Biogen Ma Inc. | Sp35 antibodies and uses thereof |
US8128926B2 (en) | 2007-01-09 | 2012-03-06 | Biogen Idec Ma Inc. | Sp35 antibodies and uses thereof |
AU2008205512B2 (en) | 2007-01-16 | 2014-06-12 | Abbvie Inc. | Methods for treating psoriasis |
WO2008092002A2 (en) | 2007-01-24 | 2008-07-31 | The Regents Of The University Of Michigan | Compositions and methods for treating and diagnosing pancreatic cancer |
EP2111553B1 (en) | 2007-01-24 | 2018-09-19 | Carnegie Mellon University | Optical biosensors |
CA2677994A1 (en) | 2007-02-07 | 2008-08-14 | Decode Genetics Ehf. | Genetic variants contributing to risk of prostate cancer |
CA2676766A1 (en) | 2007-02-09 | 2008-08-21 | Genentech, Inc. | Anti-robo4 antibodies and uses therefor |
US8685666B2 (en) * | 2007-02-16 | 2014-04-01 | The Board Of Trustees Of Southern Illinois University | ARL-1 specific antibodies and uses thereof |
WO2008101184A2 (en) * | 2007-02-16 | 2008-08-21 | The Board Of Trustees Of Southern Illinois University | Arl-1 specific antibodies |
MX2009008878A (en) | 2007-02-21 | 2009-08-28 | Decode Genetics Ehf | Genetic susceptibility variants associated with cardiovascular disease. |
ES2379413T3 (en) | 2007-02-22 | 2012-04-25 | Genentech, Inc. | Procedures to detect inflammatory bowel disease |
US7771947B2 (en) * | 2007-02-23 | 2010-08-10 | Investigen, Inc. | Methods and compositions for rapid light-activated isolation and detection of analytes |
EP2447719B1 (en) | 2007-02-26 | 2016-08-24 | Oxford BioTherapeutics Ltd | Proteins |
WO2008104803A2 (en) | 2007-02-26 | 2008-09-04 | Oxford Genome Sciences (Uk) Limited | Proteins |
US20100311767A1 (en) | 2007-02-27 | 2010-12-09 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
EP2386635A3 (en) | 2007-03-22 | 2012-02-08 | Heptares Therapeutics Limited | Mutant G-protein coupled receptors (GPCR) and methods for selecting them |
JP5721951B2 (en) | 2007-03-22 | 2015-05-20 | バイオジェン アイデック マサチューセッツ インコーポレイテッド | Binding proteins that specifically bind to CD154, including antibodies, antibody derivatives, and antibody fragments, and uses thereof |
US7960139B2 (en) | 2007-03-23 | 2011-06-14 | Academia Sinica | Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells |
RU2476442C2 (en) | 2007-03-29 | 2013-02-27 | Эббот Лэборетриз | Crystalline human il-12 antibodies |
US20110130544A1 (en) | 2007-03-30 | 2011-06-02 | Medimmune, Llc | Antibodies with decreased deamidation profiles |
EP2068909B1 (en) | 2007-03-30 | 2012-04-25 | Ambrx, Inc. | Modified fgf-21 polypeptides and their uses |
UY30994A1 (en) | 2007-04-02 | 2008-11-28 | Amgen Fremont Inc | ANTI-IGE ANTIBODIES |
ATE501280T1 (en) * | 2007-04-04 | 2011-03-15 | Chimera Biotec Gmbh | METHOD FOR DETECTING AN ANALYTE IN A BIOLOGICAL MATRIX |
US7807168B2 (en) * | 2007-04-10 | 2010-10-05 | Vaccinex, Inc. | Selection of human TNFα specific antibodies |
EP2147096B1 (en) | 2007-04-13 | 2015-03-25 | Catalyst Biosciences, Inc. | Modified factor VII polypeptides and uses thereof |
AU2008247815B2 (en) | 2007-05-02 | 2012-09-06 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
SG194368A1 (en) | 2007-05-04 | 2013-11-29 | Technophage Investigacao E Desenvolvimento Em Biotecnologia Sa | Engineered rabbit antibody variable domains and uses thereof |
CN101861168B (en) | 2007-05-07 | 2014-07-02 | 米迪缪尼有限公司 | Anti-ICOS antibodies and their use in treatment of oncology, transplantation and autoimmune disease |
SG10201503254TA (en) | 2007-05-14 | 2015-06-29 | Medimmune Llc | Methods of reducing eosinophil levels |
BRPI0721707A2 (en) | 2007-05-17 | 2013-01-15 | Genentech Inc | crystal structures of neuropillin fragments and neuropillin-antibody complexes |
US7906149B2 (en) * | 2007-05-25 | 2011-03-15 | Boval Company, L.P. | Method for treating allergic dermatitis |
EA018444B1 (en) | 2007-05-25 | 2013-08-30 | Декоуд Дженетикс Ехф. | GENETIC VARIANTS ON Chr 5p12 AND 10q26 AS MARKERS FOR USE IN BREAST CANCER RISK ASSESSMENT, DIAGNOSIS, PROGNOSIS AND TREATMENT |
US20090175847A1 (en) * | 2007-05-30 | 2009-07-09 | Abbott Laboratories | Humanized antibodies to ab (20-42) globulomer and uses thereof |
US20090232801A1 (en) * | 2007-05-30 | 2009-09-17 | Abbot Laboratories | Humanized Antibodies Which Bind To AB (1-42) Globulomer And Uses Thereof |
EP2171451A4 (en) | 2007-06-11 | 2011-12-07 | Abbott Biotech Ltd | Methods for treating juvenile idiopathic arthritis |
CA2958672A1 (en) | 2007-06-15 | 2008-12-18 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Treatment of tumors using specific anti-l1 antibody |
WO2008154700A1 (en) * | 2007-06-20 | 2008-12-24 | Phylogica Limited | Compositions and uses thereof for the treatment of acute respiratory distress syndrome (ards) and clinical disorders associated with therewith |
MX2009013816A (en) | 2007-06-21 | 2010-02-24 | Macrogenics Inc | Covalent diabodies and uses thereof. |
ES2627223T3 (en) | 2007-06-26 | 2017-07-27 | F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H | Presentation of binding agents |
DK2173379T3 (en) | 2007-07-02 | 2015-12-07 | Oncomed Pharm Inc | Compositions and methods for treatment and diagnosis of cancer |
JP2010533495A (en) | 2007-07-16 | 2010-10-28 | ジェネンテック, インコーポレイテッド | Humanized CD79b antibodies and immunoconjugates and their use |
PT2176296E (en) | 2007-07-16 | 2012-05-14 | Genentech Inc | Anti-cd79b antibodies and immunoconjugates and methods of use |
WO2009015063A2 (en) | 2007-07-23 | 2009-01-29 | Centocor | Methods and compositions for treating fibrosis related disorders using il-17 antagonists |
BRPI0814645A2 (en) * | 2007-07-25 | 2015-01-27 | Alexion Pharma Inc | METHODS AND COMPOSITIONS FOR TREATING AUTOIMMUNE DISEASE. |
US20100273722A1 (en) * | 2007-08-06 | 2010-10-28 | Yale University | Modified miniature proteins |
ES2403904T3 (en) | 2007-08-21 | 2013-05-22 | Morphosys Ag | Methods for the formation of disulfide bonds |
CN101896499B (en) * | 2007-08-30 | 2014-02-12 | 库尔Dm股份有限公司 | Compositions and methods of using proislet peptides and analogs thereof |
GB0717337D0 (en) | 2007-09-06 | 2007-10-17 | Ucb Pharma Sa | Method of treatment |
CA2964398C (en) * | 2007-09-14 | 2023-03-07 | Adimab, Llc | Rationally designed, synthetic antibody libraries and uses therefor |
US8877688B2 (en) | 2007-09-14 | 2014-11-04 | Adimab, Llc | Rationally designed, synthetic antibody libraries and uses therefor |
US8629246B2 (en) | 2007-09-26 | 2014-01-14 | Ucb Pharma S.A. | Dual specificity antibody fusions |
AR068563A1 (en) | 2007-09-26 | 2009-11-18 | Chugai Pharmaceutical Co Ltd | CONSTANT MUTANT ANTIBODY REGION |
JP5566293B2 (en) * | 2007-10-04 | 2014-08-06 | バイオノミックス リミテッド | Endothelial cell markers and uses thereof |
SI2219452T1 (en) | 2007-11-05 | 2016-03-31 | Medimmune, Llc | Methods of treating scleroderma |
MX2010005108A (en) | 2007-11-07 | 2010-05-27 | Genentech Inc | Compositions and methods for treatment of microbial disorders. |
EP3115469B1 (en) | 2007-11-19 | 2020-04-29 | Celera Corporation | Lung cancer markers and uses thereof |
JP5547083B2 (en) * | 2007-11-20 | 2014-07-09 | アンブルックス,インコーポレイテッド | Modified insulin polypeptides and their use |
EP2225275A4 (en) * | 2007-11-28 | 2013-04-03 | Medimmune Llc | Protein formulation |
US8697360B2 (en) | 2007-11-30 | 2014-04-15 | Decode Genetics Ehf. | Genetic variants on CHR 11Q and 6Q as markers for prostate and colorectal cancer predisposition |
AR069501A1 (en) * | 2007-11-30 | 2010-01-27 | Genentech Inc | ANTI-VEGF ANTIBODIES (VASCULAR ENDOTELIAL GROWTH FACTOR) |
US8426153B2 (en) | 2007-12-03 | 2013-04-23 | Carnegie Mellon University | Linked peptides fluorogenic biosensors |
GB0724051D0 (en) | 2007-12-08 | 2008-01-16 | Medical Res Council | Mutant proteins and methods for producing them |
CA2709135C (en) | 2007-12-17 | 2018-06-05 | Pfizer Limited | Treatment of interstitial cystitis |
DK2245063T3 (en) | 2007-12-18 | 2015-12-07 | Bioalliance Cv | Antibodies that recognize carbohydrate AN EPITOPE ON CD-43 AND CEA expressed CANCER CELLS AND PRACTICES BY WHICH THEY USED |
GB0724860D0 (en) | 2007-12-20 | 2008-01-30 | Heptares Therapeutics Ltd | Screening |
CA2710252C (en) | 2007-12-20 | 2017-03-28 | Xoma Technology Ltd. | Methods for the treatment of gout |
US8637026B2 (en) | 2007-12-26 | 2014-01-28 | Vaccinex, Inc. | Anti-C35 antibody combination therapies and methods |
AU2008345022B2 (en) | 2007-12-28 | 2014-11-06 | Prothena Biosciences Limited | Treatment and prophylaxis of amyloidosis |
GB0800277D0 (en) | 2008-01-08 | 2008-02-13 | Imagination Tech Ltd | Video motion compensation |
JP2011509650A (en) | 2008-01-11 | 2011-03-31 | 株式会社ジーンテクノサイエンス | Humanized anti-α9 integrin antibody and use thereof |
US20110033378A1 (en) | 2008-01-18 | 2011-02-10 | Medlmmune, Llc. | Cysteine Engineered Antibodies For Site-Specific Conjugation |
MY157403A (en) | 2008-01-31 | 2016-06-15 | Genentech Inc | Anti-cd79b antibodies and immunoconjugates and methods of use |
PT2250279E (en) | 2008-02-08 | 2016-06-03 | Medimmune Llc | Anti-ifnar1 antibodies with reduced fc ligand affinity |
GB0802474D0 (en) | 2008-02-11 | 2008-03-19 | Heptares Therapeutics Ltd | Mutant proteins and methods for selecting them |
EP2247755B1 (en) | 2008-02-14 | 2015-01-28 | Decode Genetics EHF | Susceptibility variants for lung cancer |
WO2009108652A1 (en) | 2008-02-28 | 2009-09-03 | 3M Innovative Properties Company | Antibodies to clostridium difficile spores and uses thereof |
US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
WO2009109572A2 (en) * | 2008-03-03 | 2009-09-11 | Ablynx Nv | Monovalent phage display of single variable domains |
RU2522493C2 (en) | 2008-03-04 | 2014-07-20 | Пфайзер Лимитед | Methods of treating chronic pain |
PL2265288T3 (en) | 2008-03-04 | 2017-01-31 | Labrys Biologics Inc | Methods of treating inflammatory pain |
US20110092452A1 (en) * | 2008-03-05 | 2011-04-21 | The Regents Of The University Of Michigan | Compositions and methods for diagnosing and treating pancreatic cancer |
CN101977933A (en) | 2008-03-12 | 2011-02-16 | 奥塔哥创新有限公司 | Biomarkers |
WO2009113879A1 (en) | 2008-03-12 | 2009-09-17 | Christopher Joseph Pemberton | Biomarkers |
US9873957B2 (en) * | 2008-03-13 | 2018-01-23 | Dyax Corp. | Libraries of genetic packages comprising novel HC CDR3 designs |
AU2009225797A1 (en) | 2008-03-18 | 2009-09-24 | Abbvie Inc. | Methods for treating psoriasis |
US20110020368A1 (en) | 2008-03-25 | 2011-01-27 | Nancy Hynes | Treating cancer by down-regulating frizzled-4 and/or frizzled-1 |
CA2718120A1 (en) * | 2008-03-31 | 2009-10-08 | Genentech, Inc. | Compositions and methods for treating and diagnosing asthma |
NZ588905A (en) | 2008-04-01 | 2012-08-31 | Decode Genetics Ehf | Genetic testing method for determining susceptibility to abdominal aortic aneurysm by assay for selected polymorphisms associated with the disease |
US8669349B2 (en) | 2008-04-02 | 2014-03-11 | Macrogenics, Inc. | BCR-complex-specific antibodies and methods of using same |
SG189730A1 (en) | 2008-04-02 | 2013-05-31 | Macrogenics Inc | Her2/neu-specific antibodies and methods of using same |
KR101674097B1 (en) | 2008-04-11 | 2016-11-08 | 시애틀 지네틱스, 인크. | Detection and tratment of pancreatic, ovarian and other cancers |
GB0807413D0 (en) | 2008-04-23 | 2008-05-28 | Ucb Pharma Sa | Biological products |
WO2009131256A1 (en) | 2008-04-24 | 2009-10-29 | Gene Techno Science Co., Ltd. | Humanized antibodies specific for amino acid sequence rgd of an extracellular matrix protein and the uses thereof |
WO2009132287A2 (en) | 2008-04-24 | 2009-10-29 | Dyax Corp. | Libraries of genetic packages comprising novel hc cdr1, cdr2, and cdr3 and novel lc cdr1, cdr2, and cdr3 designs |
CA3131470A1 (en) | 2008-04-25 | 2009-10-29 | Takeda Pharmaceutical Company Limited | Fc receptor binding proteins |
US20090269786A1 (en) * | 2008-04-25 | 2009-10-29 | The Board Of Trustees Of The University Of Illinois | RHO1-Gamma Amino Butyric Acid C Receptor-Specific Antibodies |
CA2722466A1 (en) | 2008-04-29 | 2009-11-05 | Tariq Ghayur | Dual variable domain immunoglobulins and uses thereof |
EP2113255A1 (en) | 2008-05-02 | 2009-11-04 | f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H. | Cytotoxic immunoglobulin |
CA2975228C (en) | 2008-05-02 | 2020-07-21 | Seattle Genetics, Inc. | Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation |
SG190596A1 (en) | 2008-05-06 | 2013-06-28 | Genentech Inc | Affinity matured crig variants |
ES2579554T3 (en) | 2008-05-09 | 2016-08-12 | Abbvie Deutschland Gmbh & Co Kg | Antibodies for the recipient of advanced glycation terminal products (RAGE) and uses thereof |
CA2723614C (en) * | 2008-05-16 | 2015-07-14 | Genentech, Inc. | Use of biomarkers for assessing treatment of gastrointestinal inflammatory disorders with beta7 integrin antagonists |
US8093018B2 (en) | 2008-05-20 | 2012-01-10 | Otsuka Pharmaceutical Co., Ltd. | Antibody identifying an antigen-bound antibody and an antigen-unbound antibody, and method for preparing the same |
EP2304439A4 (en) | 2008-05-29 | 2012-07-04 | Nuclea Biotechnologies Llc | Anti-phospho-akt antibodies |
WO2009149189A2 (en) | 2008-06-03 | 2009-12-10 | Abbott Laboratories | Dual variable domain immunoglobulins and uses thereof |
TW201006485A (en) | 2008-06-03 | 2010-02-16 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
EP2282770B1 (en) | 2008-06-04 | 2018-03-07 | MacroGenics, Inc. | Antibodies with altered binding to fcrn and methods of using same |
WO2009150623A1 (en) | 2008-06-13 | 2009-12-17 | Pfizer Inc | Treatment of chronic prostatitis |
AU2009269542A1 (en) | 2008-07-07 | 2010-01-14 | Decode Genetics Ehf | Genetic variants for breast cancer risk assessment |
TW201014602A (en) | 2008-07-08 | 2010-04-16 | Abbott Lab | Prostaglandin E2 binding proteins and uses thereof |
JP5674654B2 (en) | 2008-07-08 | 2015-02-25 | アッヴィ・インコーポレイテッド | Prostaglandin E2 double variable domain immunoglobulin and use thereof |
PL2307051T3 (en) | 2008-07-08 | 2015-07-31 | Oncomed Pharm Inc | Notch-binding agents and antagonists and methods of use thereof |
CA2729961C (en) * | 2008-07-09 | 2018-05-01 | Biogen Idec Ma Inc. | Li113, li62 variant co2, anti-lingo antibodies |
EP2318832B1 (en) | 2008-07-15 | 2013-10-09 | Academia Sinica | Glycan arrays on ptfe-like aluminum coated glass slides and related methods |
US10138283B2 (en) | 2008-07-23 | 2018-11-27 | Ambrx, Inc. | Modified bovine G-CSF polypeptides and their uses |
JP5665740B2 (en) | 2008-07-23 | 2015-02-04 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Deacetylase inhibitors and uses thereof |
US9182406B2 (en) | 2008-08-04 | 2015-11-10 | Biodesy, Inc. | Nonlinear optical detection of molecules comprising an unnatural amino acid possessing a hyperpolarizability |
AU2009279090A1 (en) | 2008-08-06 | 2010-02-11 | Novo Nordisk Health Care Ag | Conjugated proteins with prolonged in vivo efficacy |
US8652843B2 (en) | 2008-08-12 | 2014-02-18 | Oncomed Pharmaceuticals, Inc. | DDR1-binding agents and methods of use thereof |
CA2733642A1 (en) | 2008-08-14 | 2010-02-18 | Cephalon Australia Pty Ltd | Anti-il-12/il-23 antibodies |
JP5723774B2 (en) | 2008-09-05 | 2015-05-27 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Continuous directed evolution of proteins and nucleic acids |
TWI516501B (en) | 2008-09-12 | 2016-01-11 | 禮納特神經系統科學公司 | Pcsk9 antagonists |
WO2010033237A2 (en) * | 2008-09-22 | 2010-03-25 | Calmune Corporation | Methods for creating diversity in libraries and libraries, display vectors and methods, and displayed molecules |
WO2010033229A2 (en) * | 2008-09-22 | 2010-03-25 | Calmune Corporation | Methods and vectors for display of molecules and displayed molecules and collections |
EP2344180A2 (en) | 2008-09-23 | 2011-07-20 | Wyeth LLC | Methods for predicting production of activating signals by cross-linked binding proteins |
US20110287026A1 (en) | 2008-09-23 | 2011-11-24 | President And Fellows Of Harvard College | Sirt4 and uses thereof |
MX348657B (en) | 2008-09-26 | 2017-06-21 | Ambrx Inc | Non-natural amino acid replication-dependent microorganisms and vaccines. |
WO2010036856A2 (en) | 2008-09-26 | 2010-04-01 | Wyeth Llc | Compatible display vector systems |
PL2342226T3 (en) | 2008-09-26 | 2017-01-31 | Dana-Farber Cancer Institute, Inc. | Human anti-pd-1, pd-l1, and pd-l2 antibodies and uses thereof |
WO2010037041A2 (en) | 2008-09-26 | 2010-04-01 | Oncomed Pharmaceuticals, Inc. | Frizzled-binding agents and uses thereof |
CN107022020A (en) | 2008-09-26 | 2017-08-08 | Ambrx公司 | The animal erythropoietin polypeptides and its purposes of modification |
EP2340038B1 (en) | 2008-10-10 | 2018-01-10 | Children's Medical Center Corporation | Biochemically stabilized hiv-1 env trimer vaccine |
WO2010045315A1 (en) | 2008-10-14 | 2010-04-22 | Dyax Corp. | Use of igf-ii/igf-iie binding for the treatment and prevention of systemic sclerosis associated pulmonary fibrosis |
SG10201702922VA (en) | 2008-10-20 | 2017-06-29 | Abbvie Inc | Isolation and purification of antibodies using protein a affinity chromatography |
EP2360172B1 (en) | 2008-10-20 | 2017-10-18 | Gwangju Institute of Science and Technology | Bipodal peptide binder |
CA2741523C (en) | 2008-10-24 | 2022-06-21 | Jonathan S. Towner | Human ebola virus species and compositions and methods thereof |
RU2553214C2 (en) | 2008-10-29 | 2015-06-10 | Аблинкс Н.В. | Methods of purifying single-domain antigen-binding molecules |
DK3199553T3 (en) | 2008-10-29 | 2019-07-22 | Circular Commitment Company | METHODS AND AGENTS FOR DIAGNOSTICATION AND TREATMENT OF HEPATOCELLULAR CARCINOM |
MX345226B (en) | 2008-10-29 | 2017-01-20 | Ablynx Nv | Formulations of single domain antigen binding molecules. |
KR101781907B1 (en) | 2008-10-31 | 2017-09-26 | 얀센 바이오테크 인코포레이티드 | Fibronectin type iii domain based scaffold compositions, methods and uses |
DK2356270T3 (en) | 2008-11-07 | 2016-12-12 | Fabrus Llc | Combinatorial antibody libraries and uses thereof |
WO2010054403A1 (en) | 2008-11-10 | 2010-05-14 | Alexion Pharmaceuticals, Inc. | Methods and compositions for treating complement-associated disorders |
JP5716180B2 (en) | 2008-11-11 | 2015-05-13 | ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン | Anti-CXCR1 compositions and methods |
EP2358392B1 (en) | 2008-11-12 | 2019-01-09 | MedImmune, LLC | Antibody formulation |
ATE523603T1 (en) * | 2008-11-21 | 2011-09-15 | Chimera Biotec Gmbh | CONJUGATE COMPLEXES FOR ANALYTE DETECTION |
JP6041489B2 (en) | 2008-11-22 | 2016-12-07 | ジェネンテック, インコーポレイテッド | Use of anti-VEGF antibodies in combination with chemotherapy for the treatment of breast cancer |
CA2744555A1 (en) | 2008-11-26 | 2010-06-03 | Five Prime Therapeutics, Inc. | Compositions and methods for regulating collagen and smooth muscle actin expression by serpine2 |
CA2744449C (en) | 2008-11-28 | 2019-01-29 | Emory University | Methods for the treatment of infections and tumors |
SG171812A1 (en) * | 2008-12-04 | 2011-07-28 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
HUE034832T2 (en) | 2008-12-09 | 2021-12-28 | Hoffmann La Roche | Anti-pd-l1 antibodies and their use to enhance t-cell function |
SI2786762T1 (en) | 2008-12-19 | 2019-07-31 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
US20100216115A1 (en) | 2008-12-23 | 2010-08-26 | Genentech, Inc. | Methods and compositions for diagnostic use in cancer patients |
CA2748757A1 (en) | 2008-12-31 | 2010-07-08 | Biogen Idec Ma Inc. | Anti-lymphotoxin antibodies |
US9181315B2 (en) | 2009-01-08 | 2015-11-10 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for induced brown fat differentiation |
GB0900425D0 (en) | 2009-01-12 | 2009-02-11 | Ucb Pharma Sa | Biological products |
WO2010082134A1 (en) | 2009-01-14 | 2010-07-22 | Iq Therapeutics Bv | Combination antibodies for the treatment and prevention of disease caused by bacillus anthracis and related bacteria and their toxins |
KR101245929B1 (en) | 2009-01-20 | 2013-03-22 | 호메이욘 에이치. 자데흐 | Antibody mediated osseous regeneration |
AU2010207552A1 (en) | 2009-01-21 | 2011-09-01 | Oxford Biotherapeutics Ltd. | PTA089 protein |
JP5816097B2 (en) | 2009-01-22 | 2015-11-18 | ノヴォ・ノルディスク・ヘルス・ケア・アーゲー | Stable growth hormone compound |
EP2391748A4 (en) * | 2009-01-28 | 2012-08-01 | Antigen Express Inc | Li-key hybrid peptides that modulate the immune response to influenza |
PE20120586A1 (en) | 2009-01-29 | 2012-06-17 | Abbott Lab | IL-1 BINDING PROTEINS |
US20110165063A1 (en) * | 2009-01-29 | 2011-07-07 | Abbott Laboratories | Il-1 binding proteins |
WO2010086828A2 (en) | 2009-02-02 | 2010-08-05 | Rinat Neuroscience Corporation | Agonist anti-trkb monoclonal antibodies |
WO2010087927A2 (en) | 2009-02-02 | 2010-08-05 | Medimmune, Llc | Antibodies against and methods for producing vaccines for respiratory syncytial virus |
EP2396011B1 (en) | 2009-02-12 | 2016-04-13 | Janssen Biotech, Inc. | Fibronectin type iii domain based scaffold compositions, methods and uses |
WO2010093993A2 (en) | 2009-02-12 | 2010-08-19 | Human Genome Sciences, Inc. | Use of b lymphocyte stimulator protein antagonists to promote transplantation tolerance |
AU2010216152B2 (en) * | 2009-02-17 | 2015-05-14 | Ucb Biopharma Sprl | Antibody molecules having specificity for human OX40 |
CA2752854A1 (en) | 2009-02-18 | 2010-08-26 | Carnegie Mellon University | Quenched dendrimeric dyes for bright detection |
US8030026B2 (en) | 2009-02-24 | 2011-10-04 | Abbott Laboratories | Antibodies to troponin I and methods of use thereof |
AR075798A1 (en) | 2009-03-05 | 2011-04-27 | Abbott Lab | PROTEINS OF UNION TO IL-17 (INTERLEUQUINA 17) |
US20100227335A1 (en) | 2009-03-05 | 2010-09-09 | Becton, Dickinson And Company | Matrix metalloproteinase-7 (mmp-7) monoclonal antibodies and methods for their use in the detection of ovarian cancer |
CA2753713A1 (en) | 2009-03-06 | 2010-09-10 | Tripath Imaging, Inc | Glycodelin monoclonal antibodies and methods for their use in the detection of ovarian cancer |
WO2010100247A1 (en) | 2009-03-06 | 2010-09-10 | Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research | Novel therapy for anxiety |
AU2010221993C1 (en) | 2009-03-10 | 2015-07-09 | Gene Techno Science Co., Ltd. | Generation, expression and characterization of the humanized K33N monoclonal antibody |
GB0904214D0 (en) | 2009-03-11 | 2009-04-22 | Ucb Pharma Sa | Biological products |
CA2753988C (en) | 2009-03-25 | 2017-04-25 | Genentech, Inc. | Novel anti-.alpha.5.beta. integrin antibodies and uses thereof |
CN104788564A (en) | 2009-03-25 | 2015-07-22 | 健泰科生物技术公司 | Anti-FGFR3 antibodies and methods using same |
JP6132548B2 (en) | 2009-04-01 | 2017-05-24 | ジェネンテック, インコーポレイテッド | Anti-FcRH5 antibodies and immunoconjugates and methods of use |
NZ595918A (en) | 2009-04-03 | 2013-07-26 | Decode Genetics Ehf | Genetic markers for risk management of atrial fibrillation and stroke |
EP2241323A1 (en) | 2009-04-14 | 2010-10-20 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Tenascin-W and brain cancers |
EP2421898B1 (en) | 2009-04-20 | 2016-03-16 | Oxford BioTherapeutics Ltd | Antibodies specific to cadherin-17 |
EP2424567B1 (en) * | 2009-04-27 | 2018-11-21 | OncoMed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
US8636704B2 (en) | 2009-04-29 | 2014-01-28 | Abbvie Biotechnology Ltd | Automatic injection device |
EP2424891B1 (en) | 2009-04-29 | 2014-06-11 | The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. | Erg monoclonal antibodies |
CA2762837C (en) * | 2009-05-20 | 2021-08-03 | Novimmune S.A. | Synthetic polypeptide libraries and methods for generating naturally diversified polypeptide variants |
KR101224468B1 (en) | 2009-05-20 | 2013-01-23 | 주식회사 파멥신 | Bispecific antibody having a novel form and use thereof |
US8680055B2 (en) | 2009-06-03 | 2014-03-25 | University Of Southern California | Methods for decreasing steroidogenesis in prostate cancer cells |
EP2440234A4 (en) | 2009-06-10 | 2013-11-06 | Univ New York | Immunological targeting of pathological tau proteins |
EP2261242A1 (en) | 2009-06-10 | 2010-12-15 | Universite Catholique De Louvain | Aspartate-N-acetyltransferase enzyme, diagnostic method and therapeutic method |
WO2010146511A1 (en) | 2009-06-17 | 2010-12-23 | Pfizer Limited | Treatment of overactive bladder |
EP2445520A4 (en) | 2009-06-22 | 2013-03-06 | Medimmune Llc | ENGINEERED Fc REGIONS FOR SITE-SPECIFIC CONJUGATION |
GB0910725D0 (en) | 2009-06-22 | 2009-08-05 | Heptares Therapeutics Ltd | Mutant proteins and methods for producing them |
EP2272979A1 (en) | 2009-06-30 | 2011-01-12 | Centre National de la Recherche Scientifique (CNRS) | Method for testing a subject thought to be predisposed to having cancer |
CA2766861A1 (en) | 2009-07-09 | 2011-01-13 | F. Hoffmann-La Roche Ag | In vivo tumor vasculature imaging |
EP2451977A4 (en) | 2009-07-10 | 2013-01-02 | Decode Genetics Ehf | Genetic markers associated with risk of diabetes mellitus |
WO2011014457A1 (en) | 2009-07-27 | 2011-02-03 | Genentech, Inc. | Combination treatments |
US9259476B2 (en) | 2009-07-31 | 2016-02-16 | Wayne State University | Monophosphorylated lipid A derivatives |
WO2011014771A1 (en) | 2009-07-31 | 2011-02-03 | Wayne State University | Monophosphorylated lipid a derivatives |
EP2459591B1 (en) | 2009-07-31 | 2014-08-20 | Genentech, Inc. | Inhibition of tumor metastasis using anti-g-csf-antibodies |
US8841249B2 (en) | 2009-08-06 | 2014-09-23 | Novo Nordisk A/S | Growth hormones with prolonged in-vivo efficacy |
EP3431588A1 (en) | 2009-08-11 | 2019-01-23 | F. Hoffmann-La Roche AG | Production of proteins in glutamine-free cell culture media |
US8716344B2 (en) | 2009-08-11 | 2014-05-06 | President And Fellows Of Harvard College | Class- and isoform-specific HDAC inhibitors and uses thereof |
EA023179B1 (en) * | 2009-08-13 | 2016-05-31 | Круселл Холланд Б.В. | Antibodies against human respiratory syncytial virus (rsv) and methods of use |
RU2576027C2 (en) | 2009-08-15 | 2016-02-27 | Дженентек, Инк. | Anti-angiogenesis therapy for treating previously treated breast cancer |
EP2292266A1 (en) | 2009-08-27 | 2011-03-09 | Novartis Forschungsstiftung, Zweigniederlassung | Treating cancer by modulating copine III |
US8623366B2 (en) | 2009-08-28 | 2014-01-07 | Labrys Biologics, Inc. | Methods for treating visceral pain by administering antagonist antibodies directed against calcitonin gene-related peptide |
CN105131112A (en) | 2009-08-29 | 2015-12-09 | Abbvie公司 | Therapeutic dll4 binding proteins |
US20110059111A1 (en) | 2009-09-01 | 2011-03-10 | Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Center | Mammalian receptors as targets for antibody and active vaccination therapy against mold infections |
AU2010289527C1 (en) | 2009-09-01 | 2014-10-30 | Abbvie Inc. | Dual variable domain immunoglobulins and uses thereof |
JP5887270B2 (en) | 2009-09-02 | 2016-03-16 | ジェネンテック, インコーポレイテッド | Mutant SMOOTHENED AND METHOD OF USING THE SAME |
WO2011030107A1 (en) | 2009-09-10 | 2011-03-17 | Ucb Pharma S.A. | Multivalent antibodies |
AU2010291927A1 (en) | 2009-09-14 | 2012-04-12 | AbbVie Deutschland GmbH & Co. KG | Methods for treating psoriasis |
EP2478136A4 (en) * | 2009-09-14 | 2013-09-25 | Dyax Corp | Libraries of genetic packages comprising novel hc cdr3 designs |
CA2772670A1 (en) | 2009-09-17 | 2011-03-24 | F. Hoffmann-La Roche Ag | Methods and compositions for diagnostics use in cancer patients |
WO2011035205A2 (en) | 2009-09-18 | 2011-03-24 | Calmune Corporation | Antibodies against candida, collections thereof and methods of use |
EP2480573A1 (en) | 2009-09-22 | 2012-08-01 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by modulating mex-3 |
EP3187877A1 (en) | 2009-09-25 | 2017-07-05 | XOMA Technology Ltd. | Screening methods |
WO2011036555A1 (en) | 2009-09-25 | 2011-03-31 | University Of Oslo | Multivalent phage display systems and methods |
US8926976B2 (en) | 2009-09-25 | 2015-01-06 | Xoma Technology Ltd. | Modulators |
EP2480572B1 (en) | 2009-09-25 | 2019-01-30 | The United States of America, as represented by The Secretary, Department of Health and Human Services | Neutralizing antibodies to hiv-1 and their use |
GB201005063D0 (en) | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
AU2010300531A1 (en) | 2009-09-30 | 2012-05-24 | President And Fellows Of Harvard College | Methods for modulation of autophagy through the modulation of autophagy-inhibiting gene products |
UY32914A (en) | 2009-10-02 | 2011-04-29 | Sanofi Aventis | ANTIBODIES SPECIFICALLY USED TO THE EPHA2 RECEIVER |
WO2011047083A1 (en) | 2009-10-13 | 2011-04-21 | Oxford Biotherapeutics Ltd. | Antibodies against epha10 |
WO2011045352A2 (en) | 2009-10-15 | 2011-04-21 | Novartis Forschungsstiftung | Spleen tyrosine kinase and brain cancers |
EP2488658A4 (en) | 2009-10-15 | 2013-06-19 | Abbvie Inc | Dual variable domain immunoglobulins and uses thereof |
DK2488204T3 (en) | 2009-10-16 | 2016-06-06 | Oncomed Pharm Inc | Therapeutic combination and use of DLL4 antagonist antibodies and blood pressure lowering agents |
CN104744560A (en) | 2009-10-20 | 2015-07-01 | Abbvie公司 | Isolation and purification of anti-il-13 antibodies using protein affinity chromatography |
AR078716A1 (en) | 2009-10-22 | 2011-11-30 | Genentech Inc | ANTIHEPSIN ANTIBODIES AND METHODS FOR USE |
ES2564207T3 (en) | 2009-10-22 | 2016-03-18 | F. Hoffmann-La Roche Ag | Methods and compositions for modulating hepsin activation of macrophage stimulating protein |
WO2011056502A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Bone morphogenetic protein receptor type ii compositions and methods of use |
WO2011056494A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor-like kinase-1 antagonist and vegfr3 antagonist combinations |
WO2011056497A1 (en) | 2009-10-26 | 2011-05-12 | Genentech, Inc. | Activin receptor type iib compositions and methods of use |
US20110098862A1 (en) | 2009-10-27 | 2011-04-28 | ExxonMobil Research Engineering Company Law Department | Multi-stage processes and control thereof |
GB0922434D0 (en) | 2009-12-22 | 2010-02-03 | Ucb Pharma Sa | antibodies and fragments thereof |
GB0922435D0 (en) | 2009-12-22 | 2010-02-03 | Ucb Pharma Sa | Method |
US9234037B2 (en) | 2009-10-27 | 2016-01-12 | Ucb Biopharma Sprl | Method to generate antibodies to ion channels |
JP6095368B2 (en) | 2009-10-27 | 2017-03-15 | ユセベ ファルマ ソシエテ アノニム | Nav1.7 antibody whose function is modified |
UY32979A (en) | 2009-10-28 | 2011-02-28 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
US20120213801A1 (en) | 2009-10-30 | 2012-08-23 | Ekaterina Gresko | Phosphorylated Twist1 and cancer |
WO2011053707A1 (en) | 2009-10-31 | 2011-05-05 | Abbott Laboratories | Antibodies to receptor for advanced glycation end products (rage) and uses thereof |
US20120282177A1 (en) | 2009-11-02 | 2012-11-08 | Christian Rohlff | ROR1 as Therapeutic and Diagnostic Target |
JP2013509874A (en) | 2009-11-04 | 2013-03-21 | エラスムス ユニバーシティ メディカル センター ロッテルダム | Novel compounds for modulating angiogenesis and methods of treatment using these compounds |
SI2496601T1 (en) | 2009-11-05 | 2017-09-29 | F. Hoffmann-La Roche Ag | Methods and composition for secretion of heterologous polypeptides |
EP2499491B1 (en) | 2009-11-11 | 2015-04-01 | Gentian AS | Immunoassay for assessing related analytes of different origin |
CA2778953C (en) | 2009-11-13 | 2020-01-14 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for the diagnosis, prognosis, monitoring, treatment and modulation of post-transplant lymphoproliferative disorders and hypoxia associated angiogenesis disorders using galectin-1 |
GB0920127D0 (en) | 2009-11-17 | 2009-12-30 | Ucb Pharma Sa | Antibodies |
GB0920324D0 (en) | 2009-11-19 | 2010-01-06 | Ucb Pharma Sa | Antibodies |
EP3002297B1 (en) | 2009-11-30 | 2020-04-08 | F. Hoffmann-La Roche AG | Antibodies for treating and diagnosing tumors expressing slc34a2 (tat211) |
US11377485B2 (en) | 2009-12-02 | 2022-07-05 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
US10087236B2 (en) | 2009-12-02 | 2018-10-02 | Academia Sinica | Methods for modifying human antibodies by glycan engineering |
JP5951498B2 (en) | 2009-12-08 | 2016-07-13 | アッヴィ・ドイチュラント・ゲー・エム・ベー・ハー・ウント・コー・カー・ゲー | Monoclonal antibody against RGMA protein for use in the treatment of retinal nerve fiber layer degeneration |
ES2523472T3 (en) | 2009-12-09 | 2014-11-26 | Institut National De La Sante Et De La Recherche Medicale | Monoclonal antibodies that bind to B7H6 and their uses |
AR078377A1 (en) | 2009-12-11 | 2011-11-02 | Genentech Inc | ANTI-VEGF-C ANTIBODIES (ISOLATED ANTI-VASCULAR ENDOTELIAL GROWTH FACTOR C) AND ITS METHODS OF USE |
MY159156A (en) | 2009-12-21 | 2016-12-15 | Genentech Inc | Antibody formulation |
CA2784800A1 (en) | 2009-12-21 | 2011-07-21 | Ambrx, Inc. | Modified porcine somatotropin polypeptides and their uses |
AU2010341516B2 (en) | 2009-12-21 | 2014-01-16 | Ambrx, Inc. | Modified bovine somatotropin polypeptides and their uses |
TW201125583A (en) * | 2009-12-23 | 2011-08-01 | Bioalliance Cv | Anti-EpCAM antibodies that induce apoptosis of cancer cells and methods using same |
EP2516465B1 (en) | 2009-12-23 | 2016-05-18 | F.Hoffmann-La Roche Ag | Anti-bv8 antibodies and uses thereof |
CA3168591A1 (en) | 2010-01-06 | 2011-07-14 | Takeda Pharmaceutical Company Limited | Plasma kallikrein binding proteins |
RU2012134369A (en) | 2010-01-11 | 2014-02-20 | Алексион Фармасьютикалз, Инк | BIOMARKERS OF IMMUNOMODULATING EFFECTS IN PEOPLE EXPOSED TO ANTIMETAL TREATMENT AGAINST CD200 |
TWI535445B (en) | 2010-01-12 | 2016-06-01 | 安可美德藥物股份有限公司 | Wnt antagonists and methods of treatment and screening |
GB201000467D0 (en) | 2010-01-12 | 2010-02-24 | Ucb Pharma Sa | Antibodies |
EP2523682B1 (en) | 2010-01-13 | 2015-12-16 | Oncomed Pharmaceuticals, Inc. | Notch1 binding agents and methods of use thereof |
WO2011088163A1 (en) | 2010-01-14 | 2011-07-21 | President And Fellows Of Harvard College | Methods for modulating skeletal remodeling and patterning by modulating shn2 activity, shn3 activity, or shn2 and shn3 activity in combination |
AU2011208620B2 (en) | 2010-01-22 | 2015-04-16 | Novo Nordisk Health Care Ag | Stable growth hormone compounds |
CA2787685A1 (en) | 2010-01-22 | 2011-07-28 | Dana-Farber Cancer Institute, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of metabolic disorders |
AU2011208625C1 (en) | 2010-01-22 | 2022-08-18 | Novo Nordisk Health Care Ag | Growth hormones with prolonged in-vivo efficacy |
JP2013518590A (en) | 2010-02-02 | 2013-05-23 | アボツト・バイオテクノロジー・リミテツド | Methods and compositions for predicting responsiveness to treatment with a TNF-α inhibitor |
TWI518325B (en) | 2010-02-04 | 2016-01-21 | 自治醫科大學 | Identification, assessment, and therapy of cancers with innate or acquired resistance to alk inhibitors |
MX341687B (en) | 2010-02-10 | 2016-08-30 | Immunogen Inc | Cd20 antibodies and uses thereof. |
WO2011103330A2 (en) | 2010-02-17 | 2011-08-25 | The Johns Hopkins University | Novel phosphorylation of cardiac troponin i as a monitor for cardiac injury |
SI2536748T1 (en) | 2010-02-18 | 2014-12-31 | Genentech, Inc. | Neuregulin antagonists and use thereof in treating cancer |
WO2011101328A2 (en) | 2010-02-18 | 2011-08-25 | Roche Glycart Ag | Treatment with a humanized igg class anti egfr antibody and an antibody against insulin like growth factor 1 receptor |
CN103237810A (en) | 2010-02-23 | 2013-08-07 | 霍夫曼-拉罗奇有限公司 | Anti-angiogenesis therapy for the treatment of ovarian cancer |
KR20130004579A (en) | 2010-02-23 | 2013-01-11 | 제넨테크, 인크. | Compositions and methods for the diagnosis and treatment of tumor |
EP3196212B1 (en) | 2010-02-24 | 2020-06-03 | ImmunoGen, Inc. | Immunoconjugates comprising a folate receptor 1 antibody |
AR080291A1 (en) | 2010-02-24 | 2012-03-28 | Rinat Neuroscience Corp | ANTI-BODIES ANTAGONISTS ANTI RECEIVER OF IL-7 AND PROCEDURES |
RU2016146198A (en) | 2010-03-02 | 2018-12-19 | Эббви Инк. | THERAPEUTIC DLL4-BINDING PROTEINS |
WO2011107586A1 (en) | 2010-03-05 | 2011-09-09 | Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research, | Smoc1, tenascin-c and brain cancers |
SA111320266B1 (en) | 2010-03-11 | 2015-06-21 | رينات نيوروساينس كوربوريشن | Antibodies with pH Dependent Antigen Binding |
RS59389B1 (en) | 2010-03-12 | 2019-11-29 | Debiopharm Int Sa | Cd37-binding molecules and immunoconjugates thereof |
DK2547359T3 (en) | 2010-03-15 | 2016-06-06 | The Board Of Trustees Of The Univ Of Illionis | Inhibitors of beta integrin G-protein alpha subunit-BINDING INTERACTIONS |
WO2011116276A2 (en) * | 2010-03-18 | 2011-09-22 | Matthew Delisa | Engineering correctly folded antibodies using inner membrane display of twin-arginine translocation intermediates |
EP2550529B1 (en) | 2010-03-23 | 2021-11-17 | Iogenetics, LLC. | Bioinformatic processes for determination of peptide binding |
EP2550295A1 (en) | 2010-03-24 | 2013-01-30 | F. Hoffmann-La Roche AG | Anti-lrp6 antibodies |
US10472426B2 (en) | 2010-03-25 | 2019-11-12 | Ucb Biopharma Sprl | Disulfide stabilized DVD-Ig molecules |
GB201005064D0 (en) | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
CA2793959C (en) | 2010-03-25 | 2019-06-04 | Oregon Health & Science University | Cmv glycoproteins and recombinant vectors |
JP2013530929A (en) | 2010-04-01 | 2013-08-01 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Frizzled binders and uses thereof |
US10338069B2 (en) | 2010-04-12 | 2019-07-02 | Academia Sinica | Glycan arrays for high throughput screening of viruses |
ES2684475T3 (en) | 2010-04-15 | 2018-10-03 | Abbvie Inc. | Proteins that bind to beta amyloid |
US20130034543A1 (en) | 2010-04-19 | 2013-02-07 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Resear | Modulating xrn1 |
EP2380909A1 (en) | 2010-04-26 | 2011-10-26 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | PTK-7 protein involved in breast cancer |
NZ603226A (en) | 2010-04-30 | 2015-02-27 | Alexion Pharma Inc | Anti-c5a antibodies and methods for using the antibodies |
AU2011248209B2 (en) | 2010-05-03 | 2016-03-17 | University Of Rochester | Anti-glucosaminidase passive immunization for Staphylococcus aureus infections |
RU2636461C2 (en) | 2010-05-03 | 2017-11-23 | Дженентек, Инк. | Compositions and methods for diagnosis and treatment of tumour |
WO2011141823A2 (en) | 2010-05-14 | 2011-11-17 | Orega Biotech | Methods of treating and/or preventing cell proliferation disorders with il-17 antagonists |
TWI522365B (en) | 2010-05-14 | 2016-02-21 | 艾伯維有限公司 | Il-1 binding proteins |
WO2011150079A1 (en) | 2010-05-25 | 2011-12-01 | Carnegie Mellon University | Targeted probes of cellular physiology |
WO2011153243A2 (en) | 2010-06-02 | 2011-12-08 | Genentech, Inc. | Anti-angiogenesis therapy for treating gastric cancer |
SG185430A1 (en) | 2010-06-03 | 2012-12-28 | Genentech Inc | Immuno-pet imaging of antibodies and immunoconjugates and uses therefor |
US8747854B2 (en) | 2010-06-03 | 2014-06-10 | Abbvie Biotechnology Ltd. | Methods of treating moderate to severe hidradenitis suppurativa with anti-TNF-alpha antibodies |
WO2011154485A1 (en) | 2010-06-10 | 2011-12-15 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating cancer by modulating mammalian sterile 20-like kinase 3 |
US9499813B2 (en) | 2010-06-10 | 2016-11-22 | President And Fellows Of Harvard College | Systems and methods for amplification and phage display |
JP2013528818A (en) | 2010-06-16 | 2013-07-11 | アッヴィ・インコーポレイテッド | Comparison of protein samples |
WO2011159980A1 (en) | 2010-06-18 | 2011-12-22 | Genentech, Inc. | Anti-axl antibodies and methods of use |
WO2012006500A2 (en) | 2010-07-08 | 2012-01-12 | Abbott Laboratories | Monoclonal antibodies against hepatitis c virus core protein |
PL2591006T3 (en) | 2010-07-09 | 2019-10-31 | Bioverativ Therapeutics Inc | Processable single chain molecules and polypeptides made using same |
UY33492A (en) | 2010-07-09 | 2012-01-31 | Abbott Lab | IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME |
CN103097418A (en) | 2010-07-09 | 2013-05-08 | 霍夫曼-拉罗奇有限公司 | Anti-neuropilin antibodies and methods of us |
KR101896124B1 (en) | 2010-07-09 | 2018-09-07 | 얀센 백신스 앤드 프리벤션 비.브이. | Anti-human respiratory syncytial virus(rsv) antibodies and methods of use |
WO2012009705A1 (en) | 2010-07-15 | 2012-01-19 | Zyngenia, Inc. | Ang-2 binding complexes and uses thereof |
DK3336225T3 (en) | 2010-07-16 | 2020-03-30 | Adimab Llc | ANTIBODY LIBRARIES |
CA2805572A1 (en) | 2010-07-16 | 2012-01-19 | Martin Hegen | Modified single domain antigen binding molecules and uses thereof |
EP2596010B1 (en) | 2010-07-19 | 2017-04-12 | Otago Innovation Limited | Signal biomarkers |
WO2012010582A1 (en) | 2010-07-21 | 2012-01-26 | Roche Glycart Ag | Anti-cxcr5 antibodies and methods of use |
CN103269720A (en) | 2010-07-22 | 2013-08-28 | 诺沃—诺迪斯克保健股份有限公司 | Growth hormone conjugates |
EP2596026B1 (en) | 2010-07-23 | 2020-04-08 | Trustees of Boston University | Anti-despr inhibitors as therapeutics for inhibition of pathological angiogenesis and tumor cell invasiveness and for molecular imaging and targeted delivery |
WO2012015758A2 (en) | 2010-07-30 | 2012-02-02 | Saint Louis University | Methods of treating pain |
US8862410B2 (en) | 2010-08-02 | 2014-10-14 | Population Diagnostics, Inc. | Compositions and methods for discovery of causative mutations in genetic disorders |
AU2011286024B2 (en) | 2010-08-02 | 2014-08-07 | Macrogenics, Inc. | Covalent diabodies and uses thereof |
BR112013002578A2 (en) | 2010-08-03 | 2019-05-14 | Abbvie Inc. | double variable domain immunoglobins and their uses |
KR20130045914A (en) | 2010-08-03 | 2013-05-06 | 에프. 호프만-라 로슈 아게 | Chronic lymphocytic leukemia (cll) biomarkers |
WO2012019024A2 (en) | 2010-08-04 | 2012-02-09 | Immunogen, Inc. | Her3-binding molecules and immunoconjugates thereof |
BR112013002532A2 (en) | 2010-08-05 | 2016-05-31 | Hoffmann La Roche | anti-mhc antibody anti-viral cytokine fusion protein |
WO2012019061A2 (en) | 2010-08-05 | 2012-02-09 | Stem Centrx, Inc. | Novel effectors and methods of use |
EP2420250A1 (en) | 2010-08-13 | 2012-02-22 | Universitätsklinikum Münster | Anti-Syndecan-4 antibodies |
BR112013003361B8 (en) | 2010-08-13 | 2022-01-25 | Roche Glycart Ag | Antibody, composition, antibody conjugate, pharmaceutical formulation and antibody usage |
TW201209063A (en) | 2010-08-13 | 2012-03-01 | Roche Glycart Ag | Anti-tenascin-C A2 antibodies and methods of use |
MX358739B (en) | 2010-08-14 | 2018-09-03 | Abbvie Inc Star | Amyloid-beta binding proteins. |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
SG10201506443TA (en) | 2010-08-17 | 2015-10-29 | Ambrx Inc | Modified relaxin polypeptides and their uses |
CA2802278A1 (en) | 2010-08-19 | 2012-02-23 | Veit Peter Grunert | An assay for measurement of antibodies binding to a therapeutic monoclonal antibody |
AU2011291462A1 (en) | 2010-08-19 | 2013-03-14 | Zoetis Belgium S.A. | Anti-NGF antibodies and their use |
GB201014033D0 (en) | 2010-08-20 | 2010-10-06 | Ucb Pharma Sa | Biological products |
TW201211252A (en) | 2010-08-26 | 2012-03-16 | Abbott Lab | Dual variable domain immunoglobulins and uses thereof |
SG187965A1 (en) | 2010-08-27 | 2013-04-30 | Stem Centrx Inc | Notum protein modulators and methods of use |
US20120089541A1 (en) | 2010-08-31 | 2012-04-12 | Genentech, Inc. | Biomarkers and methods of treatment |
CA2810016A1 (en) | 2010-09-03 | 2012-03-08 | Stem Centrx, Inc. | Novel modulators and methods of use |
US8551479B2 (en) | 2010-09-10 | 2013-10-08 | Oncomed Pharmaceuticals, Inc. | Methods for treating melanoma |
EP2614080A1 (en) | 2010-09-10 | 2013-07-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Phosphorylated twist1 and metastasis |
CN103201003B (en) | 2010-09-20 | 2016-04-13 | Abbvie公司 | Adopt SMBC antibody purification |
AR083006A1 (en) | 2010-09-23 | 2013-01-23 | Lilly Co Eli | FORMULATIONS FOR THE STIMULATING FACTOR OF COLONIES OF GRANULOCITS (G-CSF) BOVINE AND VARIANTS OF THE SAME |
EP2446898A1 (en) | 2010-09-30 | 2012-05-02 | Laboratorios Del. Dr. Esteve, S.A. | Use of growth hormone to enhance the immune response in immunosuppressed patients |
WO2012044921A1 (en) | 2010-10-01 | 2012-04-05 | St. Jude Children's Research Hospital | Methods and compositions for typing molecular subgroups of medulloblastoma |
DK2625197T3 (en) | 2010-10-05 | 2016-10-03 | Genentech Inc | Smoothened MUTANT AND METHODS OF USING THE SAME |
WO2012052391A1 (en) | 2010-10-19 | 2012-04-26 | Glaxo Group Limited | Polypeptide with jmjd3 catalytic activity |
EA201390472A1 (en) | 2010-10-29 | 2014-02-28 | Иммьюноджен, Инк. | NEW MOLECULES CONNECTING WITH EGFR AND THEIR IMMUNOCONJUGATES |
BR112013010569A2 (en) | 2010-10-29 | 2017-07-04 | Immunogen Inc | non-antagonistic and immunoconjugated egfr binding molecules thereof |
SG190682A1 (en) | 2010-11-10 | 2013-07-31 | Genentech Inc | Methods and compositions for neural disease immunotherapy |
WO2012065937A1 (en) | 2010-11-15 | 2012-05-24 | Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute For Biomedical Research | Anti-fungal agents |
WO2012099566A1 (en) | 2010-11-17 | 2012-07-26 | Sea Lane Biotechnologies, Llc | Influenza virus neutralizing agents that mimic the binding site of an influenza neutralizing antibody |
EP2640425A2 (en) | 2010-11-18 | 2013-09-25 | Beth Israel Deaconess Medical Center, Inc. | Methods of treating obesity by inhibiting nicotinamide n-methyl transferase (nnmt) |
WO2012071436A1 (en) | 2010-11-24 | 2012-05-31 | Genentech, Inc. | Method of treating autoimmune inflammatory disorders using il-23r loss-of-function mutants |
RU2016123839A (en) | 2010-12-08 | 2018-11-30 | АббВай Стемсентркс ЭлЭлСи | New modulators and methods of their application |
US20120156194A1 (en) | 2010-12-16 | 2012-06-21 | Genentech, Inc. | Diagnosis and treatments relating to th2 inhibition |
WO2012087943A2 (en) | 2010-12-20 | 2012-06-28 | The Regents Of The University Of Michigan | Inhibitors of the epidermal growth factor receptor-heat shock protein 90 binding interaction |
UA115641C2 (en) | 2010-12-20 | 2017-11-27 | Дженентек, Інк. | DETERMINED ANTIBODY Binds Mesothelin and Immunoconjugate Containing It |
US20120275996A1 (en) | 2010-12-21 | 2012-11-01 | Abbott Laboratories | IL-1 Binding Proteins |
MY163368A (en) | 2010-12-21 | 2017-09-15 | Abbvie Inc | Il-1-alpha and -beta bispecific dual variable domain immunoglobulins and their use |
CA2825370A1 (en) | 2010-12-22 | 2012-06-28 | President And Fellows Of Harvard College | Continuous directed evolution |
EP2654790B1 (en) | 2010-12-22 | 2019-02-06 | Teva Pharmaceuticals Australia Pty Ltd | Modified antibody with improved half-life |
US20120195910A1 (en) | 2010-12-22 | 2012-08-02 | Genentech, Inc. | Anti-pcsk9 antibodies and methods of use |
JP2014504503A (en) | 2010-12-28 | 2014-02-24 | ゾーマ テクノロジー リミテッド | Cell surface display using PDZ domains |
WO2012089814A1 (en) | 2010-12-30 | 2012-07-05 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antigen binding formats for use in therapeutic treatments or diagnostic assays |
WO2012092539A2 (en) | 2010-12-31 | 2012-07-05 | Takeda Pharmaceutical Company Limited | Antibodies to dll4 and uses thereof |
WO2012093068A1 (en) | 2011-01-03 | 2012-07-12 | F. Hoffmann-La Roche Ag | A pharmaceutical composition of a complex of an anti-dig antibody and digoxigenin that is conjugated to a peptide |
GB201100282D0 (en) | 2011-01-07 | 2011-02-23 | Ucb Pharma Sa | Biological methods |
US10208349B2 (en) | 2011-01-07 | 2019-02-19 | Ucb Biopharma Sprl | Lipocalin 2 as a biomarker for IL-17 inhibitor therapy efficacy |
LT2663577T (en) | 2011-01-14 | 2017-07-25 | Ucb Biopharma Sprl | Antibody binding il-17a and il-17f |
EP2749305B1 (en) | 2011-01-24 | 2017-11-01 | AbbVie Biotechnology Ltd | Automatic injection devices having overmolded gripping surfaces |
EP3763740A1 (en) | 2011-01-26 | 2021-01-13 | Celldex Therapeutics, Inc. | Anti-kit antibodies and uses thereof |
CA2826453A1 (en) | 2011-02-03 | 2012-08-09 | Alexion Pharmaceuticals, Inc. | Use of an anti-cd200 antibody for prolonging the survival of allografts |
WO2012109238A2 (en) | 2011-02-07 | 2012-08-16 | President And Fellows Of Harvard College | Methods for increasing immune responses using agents that directly bind to and activate ire-1 |
SA112330278B1 (en) | 2011-02-18 | 2015-10-09 | ستيم سينتركس، انك. | Novel modulators and methods of use |
EP2680925B1 (en) | 2011-03-02 | 2019-11-20 | Berg LLC | Interrogatory cell-based assays and uses thereof |
CA2865487A1 (en) | 2011-03-07 | 2012-09-13 | Paula J. Bates | Predictive marker of dnmt1 inhibitor therapeutic efficacy and methods of using the marker |
US20120238730A1 (en) | 2011-03-15 | 2012-09-20 | Abbott Laboratories | Integrated approach to the isolation and purification of antibodies |
GB2503604B (en) | 2011-03-21 | 2020-04-22 | Biodesy Llc | Classification of kinase inhibitors using second harmonic optical techniques |
WO2012128810A1 (en) | 2011-03-23 | 2012-09-27 | Abbott Laboratories | Methods and systems for the analysis of protein samples |
EP3590965A1 (en) | 2011-03-29 | 2020-01-08 | Roche Glycart AG | Antibody fc variants |
WO2012131053A1 (en) | 2011-03-30 | 2012-10-04 | Ablynx Nv | Methods of treating immune disorders with single domain antibodies against tnf-alpha |
US9777332B2 (en) | 2011-03-31 | 2017-10-03 | St. Jude Children's Research Hospital | Methods and compositions for identifying minimal residual disease in acute lymphoblastic leukemia |
BR112013024717A2 (en) | 2011-04-07 | 2017-08-08 | Genentech Inc | isolated antibody, isolated nucleic acid, host cell, immunoconjugate, pharmaceutical formulation, method of treating an individual who has cancer and method of inhibiting cell proliferation in an individual |
AU2012239961A1 (en) | 2011-04-08 | 2013-10-24 | Biogen Ma Inc. | Biomarkers predictive of therapeutic responsiveness to IFNbeta and uses thereof |
WO2012142164A1 (en) | 2011-04-12 | 2012-10-18 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Human monoclonal antibodies that bind insulin-like growth factor (igf) i and ii |
WO2012142526A1 (en) | 2011-04-14 | 2012-10-18 | Modiano Jaime | Use of tumor fas expression to determine response to anti-cancer therapy |
MX338353B (en) | 2011-04-20 | 2016-04-13 | Medimmune Llc | Antibodies and other molecules that bind b7-h1 and pd-1. |
EP2699597B1 (en) | 2011-04-21 | 2016-06-01 | Garvan Institute of Medical Research | Modified variable domain molecules and methods for producing and using them b |
CA2833212C (en) | 2011-05-12 | 2020-06-09 | Genentech, Inc. | Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature peptides |
JP2014518624A (en) | 2011-05-12 | 2014-08-07 | ザ・ジョンズ・ホプキンス・ユニバーシティー | Assay reagent for neurogranin diagnostic kit |
MY175338A (en) | 2011-05-16 | 2020-06-19 | Genentech Inc | Fgfr1 agonists and methods of use |
AU2012259162C1 (en) | 2011-05-21 | 2020-05-21 | Macrogenics, Inc. | Deimmunized serum-binding domains and their use for extending serum half-life |
WO2012162561A2 (en) | 2011-05-24 | 2012-11-29 | Zyngenia, Inc. | Multivalent and monovalent multispecific complexes and their uses |
MX343659B (en) | 2011-06-02 | 2016-11-16 | Dyax Corp | Fc RECEPTOR BINDING PROTEINS. |
KR102577578B1 (en) | 2011-06-03 | 2023-09-11 | 조마 테크놀로지 리미티드 | Antibodies specific for tgf-beta |
US9181553B2 (en) | 2011-06-06 | 2015-11-10 | Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Method of treatment of breast cancers over-expressing the SHP2 signature genes |
US9561274B2 (en) | 2011-06-07 | 2017-02-07 | University Of Hawaii | Treatment and prevention of cancer with HMGB1 antagonists |
WO2012170740A2 (en) | 2011-06-07 | 2012-12-13 | University Of Hawaii | Biomarker of asbestos exposure and mesothelioma |
PL2718320T3 (en) | 2011-06-10 | 2018-06-29 | Medimmune Limited | Anti-pseudomonas psl binding molecules and uses thereof |
CA2832109C (en) | 2011-06-10 | 2021-07-06 | Oregon Health & Science University | Cmv glycoproteins and recombinant vectors |
AR086924A1 (en) | 2011-06-15 | 2014-01-29 | Hoffmann La Roche | HUMAN EPO ANTI-RECEIVER ANTIBODIES AND THE METHODS FOR USE |
KR102058185B1 (en) | 2011-06-28 | 2019-12-20 | 옥스포드 바이오테라퓨틱스 리미티드 | Antibodies to adp-ribosyl cyclase 2 |
RS55716B1 (en) | 2011-06-28 | 2017-07-31 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
CA2835242A1 (en) | 2011-06-30 | 2013-01-03 | Genentech, Inc. | Anti-c-met antibody formulations |
EP2734236A4 (en) | 2011-07-13 | 2015-04-15 | Abbvie Inc | Methods and compositions for treating asthma using anti-il-13 antibodies |
GB201112056D0 (en) | 2011-07-14 | 2011-08-31 | Univ Leuven Kath | Antibodies |
BR112014000392A2 (en) | 2011-07-14 | 2017-02-21 | Pfizer | anti-pcsk9 antibody treatment |
EP2731625B1 (en) | 2011-07-15 | 2018-04-18 | OncoMed Pharmaceuticals, Inc. | Rspo binding agents and uses thereof |
WO2013012733A1 (en) | 2011-07-15 | 2013-01-24 | Biogen Idec Ma Inc. | Heterodimeric fc regions, binding molecules comprising same, and methods relating thereto |
US20130022551A1 (en) | 2011-07-22 | 2013-01-24 | Trustees Of Boston University | DEspR ANTAGONISTS AND AGONISTS AS THERAPEUTICS |
CN103842030B (en) | 2011-08-01 | 2018-07-31 | 霍夫曼-拉罗奇有限公司 | Use the method for PD-1 axis binding antagonists and mek inhibitor treating cancer |
JP6120848B2 (en) | 2011-08-15 | 2017-04-26 | メディミューン,エルエルシー | Anti-B7-H4 antibody and use thereof |
BR112014003431A2 (en) | 2011-08-17 | 2017-06-13 | Genentech Inc | antibody, nucleic acid, host cell, method of producing an antibody, immunoconjugate, pharmaceutical formulation, pharmaceutical agent, use of the antibody, method of treating an individual who has cancer, and time-lapse method for tumor recurrence |
AU2012298884B2 (en) | 2011-08-23 | 2017-11-16 | Foundation Medicine, Inc. | Novel KIF5B-RET fusion molecules and uses thereof |
MX356947B (en) | 2011-08-23 | 2018-06-20 | Roche Glycart Ag | Bispecific antibodies specific for t-cell activating antigens and a tumor antigen and methods of use. |
US9309306B2 (en) | 2011-08-23 | 2016-04-12 | Roche Glycart Ag | Anti-MCSP antibodies |
US20130060011A1 (en) | 2011-08-23 | 2013-03-07 | Peter Bruenker | Fc-free antibodies comprising two fab fragments and methods of use |
US20130058947A1 (en) | 2011-09-02 | 2013-03-07 | Stem Centrx, Inc | Novel Modulators and Methods of Use |
WO2013034579A1 (en) | 2011-09-05 | 2013-03-14 | Rheinische Friedrich-Wilhelms-Universität Bonn | Biosynthetic gene cluster for the production of peptide/protein analogues |
JP6216317B2 (en) | 2011-09-09 | 2017-10-18 | メディミューン リミテッド | Anti-Siglec-15 antibody and use thereof |
AU2012216792A1 (en) | 2011-09-12 | 2013-03-28 | International Aids Vaccine Initiative | Immunoselection of recombinant vesicular stomatitis virus expressing HIV-1 proteins by broadly neutralizing antibodies |
WO2013039996A1 (en) | 2011-09-13 | 2013-03-21 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for brown fat induction and activity using fndc5 |
CA2846083A1 (en) | 2011-09-15 | 2013-03-21 | Genentech, Inc. | Methods of promoting differentiation |
EP2771349B1 (en) | 2011-09-16 | 2020-02-26 | Iogenetics, LLC. | Bioinformatic processes for determination of peptide binding |
WO2013043715A1 (en) | 2011-09-19 | 2013-03-28 | Genentech, Inc. | Combination treatments comprising c-met antagonists and b-raf antagonists |
EP3485903B1 (en) | 2011-09-23 | 2022-11-16 | Mereo BioPharma 5, Inc. | Vegf/dll4 binding agents and uses thereof |
SG11201401287SA (en) | 2011-10-05 | 2014-05-29 | Genentech Inc | Methods of treating liver conditions using notch2 antagonists |
AU2012318306A1 (en) | 2011-10-06 | 2014-04-24 | The Board Of Trustees Of The University Of Illinois | Myosin binding protein- C for use in methods relating to diastolic heart failure |
DK2766483T3 (en) | 2011-10-10 | 2022-04-19 | Hospital For Sick Children | PROCEDURES AND COMPOSITIONS FOR SCREENING AND TREATING DEVELOPMENTAL DISORDERS |
WO2013054320A1 (en) | 2011-10-11 | 2013-04-18 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Antibodies to carcinoembryonic antigen-related cell adhesion molecule (ceacam) |
EP2766028B1 (en) | 2011-10-14 | 2017-08-16 | F. Hoffmann-La Roche AG | Peptide inhibitors of bace1 |
US8623821B2 (en) | 2011-10-14 | 2014-01-07 | Genentech, Inc. | Zymogen activators |
ES2687951T3 (en) | 2011-10-14 | 2018-10-30 | F. Hoffmann-La Roche Ag | Anti-HtrA1 antibodies and procedures for use |
WO2013056148A2 (en) | 2011-10-15 | 2013-04-18 | Genentech, Inc. | Methods of using scd1 antagonists |
WO2013059531A1 (en) | 2011-10-20 | 2013-04-25 | Genentech, Inc. | Anti-gcgr antibodies and uses thereof |
BR112014009799A2 (en) | 2011-10-24 | 2017-06-13 | Abbvie Inc | tnf directed immunoligants |
TW201323440A (en) | 2011-10-24 | 2013-06-16 | Abbvie Inc | Immunobinders directed against sclerostin |
EP2586461A1 (en) | 2011-10-27 | 2013-05-01 | Christopher L. Parks | Viral particles derived from an enveloped virus |
TW201325613A (en) | 2011-10-28 | 2013-07-01 | Genentech Inc | Therapeutic combinations and methods of treating melanoma |
AU2012332588B2 (en) | 2011-11-01 | 2017-09-07 | Bionomics, Inc. | Methods of blocking cancer stem cell growth |
CN104053671A (en) | 2011-11-01 | 2014-09-17 | 生态学有限公司 | Antibodies and methods of treating cancer |
AU2012332590B2 (en) | 2011-11-01 | 2016-10-20 | Bionomics, Inc. | Anti-GPR49 antibodies |
AU2012332593B2 (en) | 2011-11-01 | 2016-11-17 | Bionomics, Inc. | Anti-GPR49 antibodies |
EP3643323A1 (en) | 2011-11-02 | 2020-04-29 | University Of Rochester | Anti-glucosaminidase passive immunization for staphylococcus aureus infections |
EP2773390B1 (en) | 2011-11-03 | 2020-12-30 | Tripath Imaging, Inc. | Methods and compositions for preparing samples for immunostaining |
EP2773779B1 (en) | 2011-11-04 | 2020-10-14 | Population Bio, Inc. | Methods and compositions for diagnosing, prognosing, and treating neurological conditions |
CN104136042B (en) | 2011-11-07 | 2017-08-18 | 米迪缪尼有限公司 | Use the therapeutic alliance of anti-pseudomonad Psl and PcrV binding molecules |
US20140314787A1 (en) | 2011-11-08 | 2014-10-23 | Novartis Forschungsstiftung, Zweigniederlassung, Friedrich Miescher Institute | Treatment for neurodegenerative diseases |
EP2776838A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Early diagnostic of neurodegenerative diseases |
KR20140076602A (en) | 2011-11-08 | 2014-06-20 | 화이자 인코포레이티드 | Methods of treating inflammatory disorders using anti-m-csf antibodies |
WO2013071233A1 (en) | 2011-11-10 | 2013-05-16 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Methods for detecting infectious agents and a novel virus detected thereby |
SA112330988B1 (en) | 2011-11-11 | 2015-07-22 | رينات نيوروساينس كورب. | Antibodies specific for TROP-2 and their uses |
KR102048382B1 (en) | 2011-11-11 | 2019-11-25 | 유씨비 바이오파마 에스피알엘 | Albumin binding antibodies and binding fragments thereof |
AU2012340686A1 (en) | 2011-11-21 | 2014-06-19 | Immunogen, Inc. | Method of treatment of tumors that are resistant to EGFR therapies by EGFR antibody cytotoxic agent conjugate |
RU2014124842A (en) | 2011-11-21 | 2015-12-27 | Дженентек, Инк. | CLEANING ANTI-C-MET ANTIBODIES |
AU2012340766B2 (en) | 2011-11-23 | 2018-05-10 | Medimmune, Llc | Binding molecules specific for HER3 and uses thereof |
SG11201402619VA (en) | 2011-11-23 | 2014-10-30 | Igenica Biotherapeutics Inc | Anti-cd98 antibodies and methods of use thereof |
WO2013080050A2 (en) | 2011-11-30 | 2013-06-06 | Universitaetsklinikum Erlangen | Methods and compositions for determining responsiveness to treatment with a tnf-alpha inhibitor |
EP2788024A1 (en) | 2011-12-06 | 2014-10-15 | F.Hoffmann-La Roche Ag | Antibody formulation |
EP3800200A1 (en) | 2011-12-14 | 2021-04-07 | AbbVie Deutschland GmbH & Co. KG | Composition and method for the diagnosis and treatment of iron-related disorders |
EP2791175A2 (en) | 2011-12-14 | 2014-10-22 | Abbvie Deutschland GmbH & Co. KG | Composition and method for the diagnosis and treatment of iron-related disorders |
EP2794657B1 (en) | 2011-12-19 | 2017-10-11 | Xoma (Us) Llc | Methods for treating acne |
EP2794651B1 (en) | 2011-12-22 | 2022-09-21 | F. Hoffmann-La Roche AG | Expression vector element combinations, novel production cell generation methods and their use for the recombinant production of polypeptides |
SG11201403445YA (en) | 2011-12-22 | 2014-07-30 | Hoffmann La Roche | Full length antibody display system for eukaryotic cells and its use |
US9963511B2 (en) | 2011-12-22 | 2018-05-08 | Hoffmann-La Roche Inc. | Expression vector organization, novel production cell generation methods and their use for the recombinant production of polypeptides |
WO2013093809A1 (en) | 2011-12-23 | 2013-06-27 | Pfizer Inc. | Engineered antibody constant regions for site-specific conjugation and methods and uses therefor |
AR089434A1 (en) | 2011-12-23 | 2014-08-20 | Genentech Inc | PROCEDURE TO PREPARE FORMULATIONS WITH HIGH CONCENTRATION OF PROTEINS |
WO2013102042A2 (en) | 2011-12-30 | 2013-07-04 | Abbvie Inc. | Dual specific binding proteins directed against il-13 and/or il-17 |
WO2013102825A1 (en) | 2012-01-02 | 2013-07-11 | Novartis Ag | Cdcp1 and breast cancer |
EP3663314A1 (en) | 2012-01-09 | 2020-06-10 | The Scripps Research Institute | Humanized antibodies with ultralong cdr3s |
JP6684490B2 (en) | 2012-01-09 | 2020-04-22 | ザ・スクリップス・リサーチ・インスティテュート | Ultralong complementarity determining regions and uses thereof |
SI2802606T1 (en) | 2012-01-10 | 2018-08-31 | Biogen Ma Inc. | Enhancement of transport of therapeutic molecules across the blood brain barrier |
WO2013109856A2 (en) | 2012-01-18 | 2013-07-25 | Genentech, Inc. | Methods of using fgf19 modulators |
SG10201601747XA (en) | 2012-01-18 | 2016-04-28 | Genentech Inc | Anti-LRP5 Antibodies And Methods Of Use |
GB201201332D0 (en) | 2012-01-26 | 2012-03-14 | Imp Innovations Ltd | Method |
PE20142168A1 (en) | 2012-01-27 | 2015-01-17 | AbbVie Deutschland GmbH and Co KG | COMPOSITION AND METHOD FOR THE DIAGNOSIS AND TREATMENT OF DISEASES ASSOCIATED WITH THE DEGENERATION OF NEURITES |
DK2812452T3 (en) | 2012-02-09 | 2020-06-29 | Population Bio Inc | METHODS AND COMPOSITIONS FOR SCREENING AND TREATING DEVELOPMENT DISORDERS |
CA2864092C (en) | 2012-02-10 | 2021-06-29 | Seattle Genetics, Inc. | Detection and treatment of cd30+ cancers |
JP6545959B2 (en) | 2012-02-11 | 2019-07-17 | ジェネンテック, インコーポレイテッド | R-Spondin rearrangement and method of using the same |
WO2013120554A1 (en) | 2012-02-15 | 2013-08-22 | Novo Nordisk A/S | Antibodies that bind peptidoglycan recognition protein 1 |
LT2814844T (en) | 2012-02-15 | 2017-10-25 | Novo Nordisk A/S | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (trem-1) |
ES2676031T3 (en) | 2012-02-15 | 2018-07-16 | F. Hoffmann-La Roche Ag | Affinity chromatography based on the Fc receptor |
US9550830B2 (en) | 2012-02-15 | 2017-01-24 | Novo Nordisk A/S | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1) |
GB201203051D0 (en) | 2012-02-22 | 2012-04-04 | Ucb Pharma Sa | Biological products |
GB201203071D0 (en) | 2012-02-22 | 2012-04-04 | Ucb Pharma Sa | Biological products |
PE20190658A1 (en) | 2012-02-24 | 2019-05-08 | Abbvie Stemcentrx Llc | NEW MODULATORS AND EMPLOYMENT METHODS |
DK2825558T3 (en) | 2012-03-13 | 2019-07-22 | Hoffmann La Roche | Combination therapy for the treatment of ovarian cancer |
MX2014010953A (en) | 2012-03-16 | 2014-10-13 | Hoffmann La Roche | Methods of treating melanoma with pak1 inhibitors. |
US9139863B2 (en) | 2012-03-16 | 2015-09-22 | Genentech, Inc. | Engineered conformationally-stabilized proteins |
EP2825549B1 (en) | 2012-03-16 | 2018-10-10 | F. Hoffmann-La Roche SA | Engineered conformationally-stabilized proteins |
ES2661516T3 (en) | 2012-03-20 | 2018-04-02 | Otago Innovation Limited | Biomarkers |
WO2013139956A1 (en) | 2012-03-22 | 2013-09-26 | Thrombogenics Nv | Antibodies abrogating cell binding to lactadherin |
CA2866185C (en) | 2012-03-23 | 2021-04-06 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Pathogenic phlebovirus isolates and compositions and methods of use |
BR112014024017A8 (en) | 2012-03-27 | 2017-07-25 | Genentech Inc | METHODS FOR TREATMENT OF A TYPE OF CANCER, FOR TREATMENT OF CARCINOMA, FOR SELECTING A THERAPY AND FOR QUANTIFICATION AND HER3 INHIBITORS |
CA2868161A1 (en) | 2012-03-28 | 2013-10-03 | Genentech,Inc. | Anti-hcmv idiotypic antibodies and uses thereof |
EP2831112A1 (en) | 2012-03-29 | 2015-02-04 | Friedrich Miescher Institute for Biomedical Research | Inhibition of interleukin- 8 and/or its receptor cxcrl in the treatment her2/her3 -overexpressing breast cancer |
AR090549A1 (en) | 2012-03-30 | 2014-11-19 | Genentech Inc | ANTI-LGR5 AND IMMUNOCATE PLAYERS |
SG10201608234UA (en) | 2012-04-02 | 2016-11-29 | Berg Llc | Interrogatory cell-based assays and uses thereof |
WO2013151649A1 (en) | 2012-04-04 | 2013-10-10 | Sialix Inc | Glycan-interacting compounds |
US10130714B2 (en) | 2012-04-14 | 2018-11-20 | Academia Sinica | Enhanced anti-influenza agents conjugated with anti-inflammatory activity |
WO2013158273A1 (en) | 2012-04-20 | 2013-10-24 | Abbvie Inc. | Methods to modulate c-terminal lysine variant distribution |
KR102382304B1 (en) | 2012-04-20 | 2022-04-04 | 메뤼스 엔.페. | Methods and means for the production of ig-like molecules |
WO2013158275A1 (en) | 2012-04-20 | 2013-10-24 | Abbvie Inc. | Cell culture methods to reduce acidic species |
US9334319B2 (en) | 2012-04-20 | 2016-05-10 | Abbvie Inc. | Low acidic species compositions |
AU2013254690B2 (en) | 2012-04-24 | 2017-12-07 | Thrombogenics N.V. | Anti-PDGF-C antibodies |
JP6302460B2 (en) | 2012-04-25 | 2018-03-28 | バイオデシー, インコーポレイテッド | Methods for detecting allosteric modulators of proteins |
CN104470544B (en) | 2012-05-01 | 2018-01-12 | 基因泰克公司 | Anti- PMEL17 antibody and immunoconjugates |
WO2013170191A1 (en) | 2012-05-11 | 2013-11-14 | Genentech, Inc. | Methods of using antagonists of nad biosynthesis from nicotinamide |
NZ702178A (en) | 2012-05-14 | 2017-01-27 | Biogen Ma Inc | Lingo-2 antagonists for treatment of conditions involving motor neurons |
JP6122948B2 (en) | 2012-05-15 | 2017-04-26 | モルフォテック, インコーポレイテッド | Methods for the treatment of gastric cancer |
WO2013177118A2 (en) | 2012-05-21 | 2013-11-28 | Abbvie Inc. | Novel purification of non-human antibodies using protein a affinity chromatography |
RU2625771C2 (en) | 2012-05-23 | 2017-07-18 | Дженентек, Инк. | Therapeutics selection method |
WO2013176754A1 (en) | 2012-05-24 | 2013-11-28 | Abbvie Inc. | Novel purification of antibodies using hydrophobic interaction chromatography |
RU2689760C2 (en) | 2012-05-31 | 2019-05-30 | Дженентек, Инк. | Methods of treating cancer using pd-1 axis binding and vegf antagonists |
US9617334B2 (en) | 2012-06-06 | 2017-04-11 | Zoetis Services Llc | Caninized anti-NGF antibodies and methods thereof |
AU2013271515A1 (en) | 2012-06-06 | 2015-01-15 | Oncomed Pharmaceuticals, Inc. | Binding agents that modulate the Hippo pathway and uses thereof |
TW201410706A (en) | 2012-06-15 | 2014-03-16 | Genentech Inc | Anti-PCSK9 antibodies, formulations, dosing, and methods of use |
EP2679596B1 (en) | 2012-06-27 | 2017-04-12 | International Aids Vaccine Initiative | HIV-1 env glycoprotein variant |
EP2867674B1 (en) | 2012-06-28 | 2018-10-10 | UCB Biopharma SPRL | A method for identifying compounds of therapeutic interest |
EP2866831A1 (en) | 2012-06-29 | 2015-05-06 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Treating diseases by modulating a specific isoform of mkl1 |
KR20150030755A (en) | 2012-07-04 | 2015-03-20 | 에프. 호프만-라 로슈 아게 | Anti-biotin antibodies and methods of use |
HUE029435T2 (en) | 2012-07-04 | 2017-02-28 | Hoffmann La Roche | Anti-theophylline antibodies and methods of use |
WO2014006124A1 (en) | 2012-07-04 | 2014-01-09 | F. Hoffmann-La Roche Ag | Covalently linked antigen-antibody conjugates |
CA3188124A1 (en) | 2012-07-05 | 2014-01-09 | Genentech, Inc. | Expression and secretion system |
EP2870242A1 (en) | 2012-07-05 | 2015-05-13 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | New treatment for neurodegenerative diseases |
US20150224190A1 (en) | 2012-07-06 | 2015-08-13 | Mohamed Bentires-Alj | Combination of a phosphoinositide 3-kinase inhibitor and an inhibitor of the IL-8/CXCR interaction |
CA2874904A1 (en) | 2012-07-09 | 2014-01-16 | Genentech, Inc. | Immunoconjugates comprising anti-cd22 antibodies |
AR091700A1 (en) | 2012-07-09 | 2015-02-25 | Genentech Inc | ANTI-CD79B ANTIBODIES AND IMMUNOCATION |
EP2869847B1 (en) | 2012-07-09 | 2017-12-06 | Genentech, Inc. | Immunoconjugates comprising anti-cd79b antibodies |
SG11201500087VA (en) | 2012-07-09 | 2015-02-27 | Genentech Inc | Immunoconjugates comprising anti-cd22 antibodies |
WO2014011955A2 (en) | 2012-07-12 | 2014-01-16 | Abbvie, Inc. | Il-1 binding proteins |
WO2014009465A1 (en) | 2012-07-13 | 2014-01-16 | Roche Glycart Ag | Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases |
GB201213652D0 (en) | 2012-08-01 | 2012-09-12 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
US9297806B2 (en) | 2012-08-01 | 2016-03-29 | The Johns Hopkins University | 5-hydroxymethylcytosine in human cancer |
WO2014022759A1 (en) | 2012-08-03 | 2014-02-06 | Dana-Farber Cancer Institute, Inc. | Agents that modulate immune cell activation and methods of use thereof |
WO2014025813A1 (en) | 2012-08-07 | 2014-02-13 | Genentech, Inc. | Combination therapy for the treatment of glioblastoma |
US9914956B2 (en) | 2012-08-18 | 2018-03-13 | Academia Sinica | Cell-permeable probes for identification and imaging of sialidases |
KR20200079565A (en) | 2012-08-31 | 2020-07-03 | 이뮤노젠 아이엔씨 | Diagnostic assays and kits for detection of folate receptor 1 |
WO2014039860A2 (en) | 2012-09-07 | 2014-03-13 | University Of Louisville Research Foundation, Inc. | Compositions and methods for modulating dnmt1 inhibitor activity |
EP2895621B1 (en) | 2012-09-14 | 2020-10-21 | Population Bio, Inc. | Methods and compositions for diagnosing, prognosing, and treating neurological conditions |
EP2900835A4 (en) | 2012-09-27 | 2016-05-11 | Population Diagnotics Inc | Methods and compositions for screening and treating developmental disorders |
AR092745A1 (en) | 2012-10-01 | 2015-04-29 | Univ Pennsylvania | COMPOSITIONS THAT INCLUDE AN ANTI-FAP UNION DOMAIN AND METHODS TO MAKE WHITE IN STROMAL CELLS FOR THE TREATMENT OF CANCER |
WO2014056783A1 (en) | 2012-10-08 | 2014-04-17 | Roche Glycart Ag | Fc-free antibodies comprising two fab-fragments and methods of use |
AU2013329421A1 (en) | 2012-10-09 | 2015-04-30 | Biogen Ma Inc. | Combination therapies and uses for treatment of demyelinating disorders |
AU2013329311A1 (en) | 2012-10-09 | 2015-04-30 | Igenica Biotherapeutics, Inc. | Anti-C16orf54 antibodies and methods of use thereof |
KR102229873B1 (en) | 2012-10-12 | 2021-03-19 | 더 브리검 앤드 우먼즈 하스피털, 인크. | Enhancement of the immune response |
CA2887711A1 (en) | 2012-10-23 | 2014-05-01 | Oncomed Pharmaceuticals, Inc. | Methods of treating neuroendocrine tumors using wnt pathway-binding agents |
JP6371294B2 (en) | 2012-10-31 | 2018-08-08 | オンコメッド ファーマシューティカルズ インコーポレイテッド | Methods and monitoring of treatment with DLL4 antagonists |
RU2017137740A (en) | 2012-11-01 | 2019-02-11 | Эббви Инк. | ANTI-VEGF / DLL4-IMMUNOGLOBULINS WITH DOUBLE VARIABLE DOMAINS AND THEIR APPLICATIONS |
EP2914621B1 (en) | 2012-11-05 | 2023-06-07 | Foundation Medicine, Inc. | Novel ntrk1 fusion molecules and uses thereof |
AU2013337354A1 (en) | 2012-11-05 | 2015-05-21 | Genzyme Corporation | Compositions and methods for treating proteinopathies |
SG11201502538TA (en) | 2012-11-08 | 2015-05-28 | Hoffmann La Roche | Her3 antigen binding proteins binding to the beta-hairpin of her3 |
CA2886987C (en) | 2012-11-08 | 2022-07-12 | Eleven Biotherapeutics, Inc. | Il-6 antagonists and uses thereof |
WO2014074942A1 (en) | 2012-11-08 | 2014-05-15 | Illumina, Inc. | Risk variants of alzheimer's disease |
CA2890669A1 (en) | 2012-11-13 | 2014-05-22 | Genetech, Inc. | Anti-hemagglutinin antibodies and methods of use |
US10407503B2 (en) | 2012-11-30 | 2019-09-10 | The Regents Of The University Of California | Fully human antibodies and fragments recognizing human c-Met |
EP2928923B1 (en) | 2012-12-10 | 2020-01-22 | Biogen MA Inc. | Anti-blood dendritic cell antigen 2 antibodies and uses thereof |
CN105102455B (en) | 2012-12-21 | 2018-05-25 | 荷商台医(有限合伙)公司 | Hydrophily consumes connexon and its conjugate certainly |
US9550986B2 (en) | 2012-12-21 | 2017-01-24 | Abbvie Inc. | High-throughput antibody humanization |
MX2015008117A (en) | 2012-12-21 | 2016-03-31 | Amplimmune Inc | Anti-h7cr antibodies. |
GB201223276D0 (en) | 2012-12-21 | 2013-02-06 | Ucb Pharma Sa | Antibodies and methods of producing same |
AU2013202668B2 (en) | 2012-12-24 | 2014-12-18 | Adelaide Research & Innovation Pty Ltd | Inhibition of cancer growth and metastasis |
WO2014107739A1 (en) | 2013-01-07 | 2014-07-10 | Eleven Biotherapeutics, Inc. | Antibodies against pcsk9 |
US10717965B2 (en) | 2013-01-10 | 2020-07-21 | Gloriana Therapeutics, Inc. | Mammalian cell culture-produced neublastin antibodies |
WO2014113729A2 (en) | 2013-01-18 | 2014-07-24 | Foundation Mecicine, Inc. | Methods of treating cholangiocarcinoma |
WO2014116749A1 (en) | 2013-01-23 | 2014-07-31 | Genentech, Inc. | Anti-hcv antibodies and methods of using thereof |
CN104955480A (en) | 2013-01-25 | 2015-09-30 | 西蒙有限公司 | Compositions for selective reduction of circulating bioactive soluble TNF and methods for treating TNF-mediated disease |
EP2950885B1 (en) | 2013-02-04 | 2018-11-21 | Oncomed Pharmaceuticals, Inc. | Methods and monitoring of treatment with a wnt pathway inhibitor |
GB201302447D0 (en) | 2013-02-12 | 2013-03-27 | Oxford Biotherapeutics Ltd | Therapeutic and diagnostic target |
WO2014129895A1 (en) | 2013-02-19 | 2014-08-28 | Stichting Vu-Vumc | Means and method for increasing the sensitivity of cancers for radiotherapy |
JP6462591B2 (en) | 2013-02-22 | 2019-01-30 | アッヴィ・ステムセントルクス・エル・エル・シー | Novel antibody conjugates and uses thereof |
CN104994879A (en) | 2013-02-22 | 2015-10-21 | 霍夫曼-拉罗奇有限公司 | Methods of treating cancer and preventing drug resistance |
RU2015140573A (en) | 2013-02-25 | 2017-03-30 | Дженентек, Инк. | METHODS AND COMPOSITIONS FOR DETECTION AND TREATMENT OF DRUG-RESISTANT MUTANT RESISTANT TO MEDICINES |
US20140242083A1 (en) | 2013-02-26 | 2014-08-28 | Roche Glycart Ag | Anti-mcsp antibodies |
BR112015021423A2 (en) | 2013-03-06 | 2017-07-18 | Genentech Inc | cancer treatment methods, egfr antagonist-resistant cancer cells, cancer cells, methods of increasing the sensitivity and effectiveness of cancer treatment, and methods of delaying, treating cancer patients and extending |
US20160017017A1 (en) * | 2013-03-11 | 2016-01-21 | Novo Nordisk Healthcare Ag | Growth Hormone Compounds |
CN105229035A (en) * | 2013-03-11 | 2016-01-06 | 诺和诺德保健股份有限公司 | Growth hormone compound |
AR095396A1 (en) | 2013-03-13 | 2015-10-14 | Genentech Inc | ANTIBODY FORMULATIONS |
AR095399A1 (en) | 2013-03-13 | 2015-10-14 | Genentech Inc | FORMULATIONS WITH REDUCED OXIDATION, METHOD |
RU2707550C2 (en) | 2013-03-13 | 2019-11-27 | Дженентек, Инк. | Compositions with reduced oxidation |
US20140314778A1 (en) | 2013-03-13 | 2014-10-23 | Genentech, Inc. | Formulations with reduced oxidation |
LT2968466T (en) | 2013-03-13 | 2018-10-10 | F. Hoffmann-La Roche Ag | Formulations with reduced oxidation |
JP2016520527A (en) | 2013-03-14 | 2016-07-14 | パーカシュ ギル, | Treatment of cancer using antibodies that bind to cell surface GRP78 |
CA2899449A1 (en) | 2013-03-14 | 2014-10-02 | Abbvie Inc. | Low acidic species compositions and methods for producing the same using displacement chromatography |
BR112015022019A2 (en) | 2013-03-14 | 2017-08-29 | Genentech Inc | ISOLATED ANTIBODIES, NUCLEIC ACID, HOST CELL, METHOD OF PRODUCING ANTIBODIES, IMMUNOCONJUGATE, PHARMACEUTICAL FORMULATION, METHODS FOR TREATMENT OF INDIVIDUALS, INHIBITION OF CELL PROLIFERATION, DETECTION OF HUMAN B7-H4 AND CANCER DETECTION |
WO2014142882A1 (en) | 2013-03-14 | 2014-09-18 | Abbvie Inc. | Protein purification using displacement chromatography |
RU2015139054A (en) | 2013-03-14 | 2017-04-19 | Дженентек, Инк. | METHODS FOR TREATING CANCER AND PREVENTION OF DRUG RESISTANCE OF CANCER |
AU2014239903A1 (en) | 2013-03-14 | 2015-09-17 | Genentech, Inc. | Combinations of a MEK inhibitor compound with an HER3/EGFR inhibitor compound and methods of use |
US9562099B2 (en) | 2013-03-14 | 2017-02-07 | Genentech, Inc. | Anti-B7-H4 antibodies and immunoconjugates |
WO2014158231A1 (en) | 2013-03-14 | 2014-10-02 | Abbvie Inc. | Low acidic species compositions and methods for producing and using the same |
US9168300B2 (en) | 2013-03-14 | 2015-10-27 | Oncomed Pharmaceuticals, Inc. | MET-binding agents and uses thereof |
CA2906417C (en) | 2013-03-14 | 2022-06-21 | Robert Ziemann | Hcv core lipid binding domain monoclonal antibodies |
CN105209616A (en) | 2013-03-14 | 2015-12-30 | 雅培制药有限公司 | HCV NS3 recombinant antigens and mutants thereof for improved antibody detection |
MX362075B (en) | 2013-03-14 | 2019-01-07 | Abbott Lab | Hcv antigen-antibody combination assay and methods and compositions for use therein. |
SG10201800313UA (en) | 2013-03-15 | 2018-02-27 | Abbvie Inc | Antibody drug conjugate (adc) purification |
EP2970459A2 (en) | 2013-03-15 | 2016-01-20 | AbbVie Inc. | Dual specific binding proteins directed against il-1beta and il-17 |
CA2903587C (en) | 2013-03-15 | 2021-09-28 | Genentech, Inc. | Il-22 polypeptides and il-22 fc fusion proteins and methods of use |
TW201945032A (en) | 2013-03-15 | 2019-12-01 | 德商艾伯維德國有限及兩合公司 | Anti-EGFR antibody drug conjugate formulations |
CN105143258B (en) | 2013-03-15 | 2020-06-23 | Ac免疫有限公司 | anti-Tau antibodies and methods of use |
SG11201507367PA (en) | 2013-03-15 | 2015-10-29 | Genentech Inc | Cell culture compositions with antioxidants and methods for polypeptide production |
CN105392801A (en) | 2013-03-15 | 2016-03-09 | 比奥根Ma公司 | Treatment and prevention of acute kidney injury using anti-alpha v beta 5 antibodies |
TWI828269B (en) | 2013-03-15 | 2024-01-01 | 美商百歐維拉提夫治療公司 | Factor ix polypeptide formulations |
SG10201706210WA (en) | 2013-03-15 | 2017-09-28 | Genentech Inc | Compositions and methods for diagnosis and treatment of hepatic cancers |
WO2014144865A2 (en) | 2013-03-15 | 2014-09-18 | Genentech, Inc. | Anti-crth2 antibodies and methods of use |
MY176706A (en) | 2013-03-15 | 2020-08-19 | Genentech Inc | Biomarkers and methods of treating pd-1 and pd-l1 related conditions |
CA2907181C (en) | 2013-03-15 | 2023-10-17 | Viktor Roschke | Multivalent and monovalent multispecific complexes and their uses |
US10160797B2 (en) | 2013-03-15 | 2018-12-25 | Sanofi Pasteur Biologics, Llc | Antibodies against Clostridium difficile toxins and methods of using the same |
KR20150130451A (en) | 2013-03-15 | 2015-11-23 | 제넨테크, 인크. | Methods of treating cancer and preventing cancer drug resistance |
RU2015144020A (en) | 2013-03-15 | 2017-04-21 | Дженентек, Инк. | ENVIRONMENTS FOR CULTIVATION OF CELLS AND METHODS FOR PRODUCING ANTIBODIES |
US9469686B2 (en) | 2013-03-15 | 2016-10-18 | Abbott Laboratories | Anti-GP73 monoclonal antibodies and methods of obtaining the same |
CN105120887A (en) | 2013-04-05 | 2015-12-02 | 诺和诺德保健股份有限公司 | Growth hormone compound formulation |
CA2944989A1 (en) | 2013-04-08 | 2014-10-16 | Cytodyn Inc. | Felinized antibodies and methods of treating retroviral infections in felines |
JP2016528168A (en) | 2013-04-29 | 2016-09-15 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | Anti-IGF-1R antibodies with ineffective FcRn binding and their use in the treatment of vascular ocular diseases |
CA2904806C (en) | 2013-04-29 | 2021-11-23 | F. Hoffmann-La Roche Ag | Human fcrn-binding modified antibodies and methods of use |
EP2992010B1 (en) | 2013-04-29 | 2021-03-24 | F.Hoffmann-La Roche Ag | Fc-receptor binding modified asymmetric antibodies and methods of use |
WO2014176696A1 (en) | 2013-04-30 | 2014-11-06 | Université de Montréal | Novel biomarkers for acute myeloid leukemia |
AU2014262843B2 (en) | 2013-05-06 | 2017-06-22 | Scholar Rock, Inc. | Compositions and methods for growth factor modulation |
CA2908743A1 (en) | 2013-05-20 | 2014-11-27 | Genentech, Inc. | Anti-transferrin receptor antibodies and methods of use |
BR112015029395A2 (en) | 2013-05-24 | 2017-09-19 | Medimmune Llc | ANTI-B7-H5 ANTIBODIES AND THEIR USES |
EP3004877A4 (en) | 2013-06-06 | 2017-04-19 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for identification, assessment prevention, and treatment of cancer using pd-l1 isoforms |
NZ714765A (en) | 2013-06-06 | 2021-12-24 | Pf Medicament | Anti-c10orf54 antibodies and uses thereof |
EP3632467B1 (en) | 2013-06-07 | 2023-09-27 | Duke University | Inhibitors of complement factor h |
KR20160034893A (en) | 2013-06-13 | 2016-03-30 | 패스트 포워드 파머슈티컬스 비.브이. | CD40 signalling inhibitor and a further compound, wherein the further compound is a bile acid, a bile acid derivative, an TGR5-receptor agonist, an FXR agonist or a combination thereof, for the treatment of chronic inflammation, and the prevention of gastrointestinal cancer or fibrosis |
US10086054B2 (en) | 2013-06-26 | 2018-10-02 | Academia Sinica | RM2 antigens and use thereof |
US9981030B2 (en) | 2013-06-27 | 2018-05-29 | Academia Sinica | Glycan conjugates and use thereof |
RS60826B1 (en) | 2013-07-16 | 2020-10-30 | Hoffmann La Roche | Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors |
EP3022221B1 (en) | 2013-07-18 | 2021-09-15 | Taurus Biosciences, LLC | Humanized antibodies with ultralong complementarity determining regions |
US20160168231A1 (en) | 2013-07-18 | 2016-06-16 | Fabrus, Inc. | Antibodies with ultralong complementarity determining regions |
CA2919477A1 (en) | 2013-07-31 | 2015-02-05 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for modulating thermogenesis using pth-related and egf-related molecules |
PE20211093A1 (en) | 2013-08-02 | 2021-06-14 | Pfizer | ANTI-CXCR4 ANTIBODIES AND ANTIBODY AND DRUG CONJUGATES |
EP3030902B1 (en) | 2013-08-07 | 2019-09-25 | Friedrich Miescher Institute for Biomedical Research | New screening method for the treatment friedreich's ataxia |
EP3036320B2 (en) | 2013-08-19 | 2024-04-03 | Biogen MA Inc. | Control of protein glycosylation by culture medium supplementation and cell culture process parameters |
HUE056325T2 (en) | 2013-08-26 | 2022-02-28 | Biontech Res And Development Inc | Nucleic acids encoding human antibodies to sialyl-lewis a |
BR112016004242A8 (en) | 2013-08-28 | 2018-06-12 | Stemcentrx Inc | METHODS FOR SITE-SPECIFIC CONJUGATION OF ANTIBODIES AND COMPOSITIONS |
JP2016538318A (en) | 2013-08-28 | 2016-12-08 | ステムセントリックス, インコーポレイテッド | New SEZ6 modulator and method of use |
US10456470B2 (en) | 2013-08-30 | 2019-10-29 | Genentech, Inc. | Diagnostic methods and compositions for treatment of glioblastoma |
US10617755B2 (en) | 2013-08-30 | 2020-04-14 | Genentech, Inc. | Combination therapy for the treatment of glioblastoma |
HUE055856T2 (en) | 2013-08-30 | 2021-12-28 | Immunogen Inc | Antibodies and assays for detection of folate receptor 1 |
US20150065381A1 (en) | 2013-09-05 | 2015-03-05 | International Aids Vaccine Initiative | Methods of identifying novel hiv-1 immunogens |
US9782476B2 (en) | 2013-09-06 | 2017-10-10 | Academia Sinica | Human iNKT cell activation using glycolipids with altered glycosyl groups |
AR097685A1 (en) | 2013-09-17 | 2016-04-06 | Genentech Inc | METHODS OF USE OF ANTI-LGR5 ANTIBODIES |
WO2015048330A2 (en) | 2013-09-25 | 2015-04-02 | Biogen Idec Ma Inc. | On-column viral inactivation methods |
EP3521431A1 (en) | 2013-09-25 | 2019-08-07 | Cornell University | Compounds for inducing anti-tumor immunity and methods thereof |
EP3049442A4 (en) | 2013-09-26 | 2017-06-28 | Costim Pharmaceuticals Inc. | Methods for treating hematologic cancers |
SI3049441T1 (en) | 2013-09-27 | 2020-03-31 | F. Hoffmann-La Roche Ag | Anti-pdl1 antibody formulations |
WO2015044083A1 (en) | 2013-09-27 | 2015-04-02 | F. Hoffmann-La Roche Ag | Thermus thermophilus slyd fkbp domain specific antibodies |
EP2873423B1 (en) | 2013-10-07 | 2017-05-31 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
KR102115217B1 (en) | 2013-10-08 | 2020-05-26 | 이뮤노젠 아이엔씨 | Anti-folr1 immunoconjugate dosing regimens |
RU2016117978A (en) | 2013-10-11 | 2017-11-17 | Дженентек, Инк. | NSP4 INHIBITORS AND WAYS OF THEIR APPLICATION |
WO2015057939A1 (en) | 2013-10-18 | 2015-04-23 | Biogen Idec Ma Inc. | Anti-s1p4 antibodies and uses thereof |
US20150147333A1 (en) | 2013-10-18 | 2015-05-28 | Genentech, Inc. | Anti-rspo antibodies and methods of use |
JP6715767B2 (en) | 2013-10-23 | 2020-07-01 | ジェネンテック, インコーポレイテッド | Method for diagnosing and treating eosinophilic disease |
WO2015066027A2 (en) | 2013-10-28 | 2015-05-07 | Dots Devices, Inc. | Allergen detection |
EP3062884B1 (en) | 2013-10-29 | 2020-12-02 | President and Fellows of Harvard College | Compositions for use in the treatment of retinitis pigmentosa |
EP3066121A1 (en) | 2013-11-07 | 2016-09-14 | AbbVie Inc. | Isolation and purification of dvd-igs |
EP2891722B1 (en) | 2013-11-12 | 2018-10-10 | Population Bio, Inc. | Methods and compositions for diagnosing, prognosing, and treating endometriosis |
CA2928014A1 (en) | 2013-11-15 | 2015-05-21 | Institut Pasteur | Method for detecting a molecular marker of plasmodium falciparum artemisinin resistance |
SI3071597T1 (en) | 2013-11-21 | 2020-11-30 | F. Hoffmann-La Roche Ag | Anti-alpha-synuclein antibodies and methods of use |
CN105744955B (en) | 2013-11-25 | 2020-03-20 | 希凯姆生物治疗有限公司 | Compositions comprising anti-CEACAM 1 antibodies and anti-PD antibodies for cancer treatment |
US20160297875A1 (en) | 2013-12-07 | 2016-10-13 | Case Western Reserve University | Compositions and methods of treating thrombosis |
US20160333063A1 (en) | 2013-12-13 | 2016-11-17 | The General Hospital Corporation | Soluble high molecular weight (hmw) tau species and applications thereof |
ES2754209T3 (en) | 2013-12-13 | 2020-04-16 | Stora Enso Oyj | Multilayer cardboard |
KR20160098328A (en) | 2013-12-13 | 2016-08-18 | 제넨테크, 인크. | Anti-cd33 antibodies and immunoconjugates |
US8986694B1 (en) | 2014-07-15 | 2015-03-24 | Kymab Limited | Targeting human nav1.7 variants for treatment of pain |
KR20240017102A (en) | 2013-12-17 | 2024-02-06 | 제넨테크, 인크. | Methods of treating cancers using pd-1 axis binding antagonists and taxanes |
PE20210107A1 (en) | 2013-12-17 | 2021-01-19 | Genentech Inc | ANTI-CD3 ANTIBODIES AND METHODS OF USE |
US9045545B1 (en) | 2014-07-15 | 2015-06-02 | Kymab Limited | Precision medicine by targeting PD-L1 variants for treatment of cancer |
US8992927B1 (en) | 2014-07-15 | 2015-03-31 | Kymab Limited | Targeting human NAV1.7 variants for treatment of pain |
US9914769B2 (en) | 2014-07-15 | 2018-03-13 | Kymab Limited | Precision medicine for cholesterol treatment |
CA2933881A1 (en) | 2013-12-17 | 2015-06-25 | Genentech, Inc. | Methods of treating cancer using pd-1 axis binding antagonists and an anti-cd20 antibody |
US9067998B1 (en) | 2014-07-15 | 2015-06-30 | Kymab Limited | Targeting PD-1 variants for treatment of cancer |
KR20160099092A (en) | 2013-12-17 | 2016-08-19 | 제넨테크, 인크. | Combination therapy comprising ox40 binding agonists and pd-1 axis binding antagonists |
US10640569B2 (en) | 2013-12-19 | 2020-05-05 | Novartis Ag | Human mesothelin chimeric antigen receptors and uses thereof |
US11708411B2 (en) | 2013-12-20 | 2023-07-25 | Wake Forest University Health Sciences | Methods and compositions for increasing protective antibody levels induced by pneumococcal polysaccharide vaccines |
WO2015095809A1 (en) | 2013-12-20 | 2015-06-25 | Biogen Idec Ma Inc. | Use of perfusion seed cultures to improve biopharmaceutical fed-batch production capacity and product quality |
TWI728373B (en) | 2013-12-23 | 2021-05-21 | 美商建南德克公司 | Antibodies and methods of use |
EP2960252A1 (en) | 2014-06-26 | 2015-12-30 | Institut Pasteur | Phospholipase for treatment of immunosuppression |
ES2920677T3 (en) | 2013-12-24 | 2022-08-08 | Janssen Pharmaceutica Nv | Anti-VISTA Antibodies and Fragments |
CA2930046A1 (en) | 2014-01-03 | 2015-07-09 | F. Hoffmann-La Roche Ag | Covalently linked polypeptide toxin-antibody conjugates |
WO2015103549A1 (en) | 2014-01-03 | 2015-07-09 | The United States Of America, As Represented By The Secretary Department Of Health And Human Services | Neutralizing antibodies to hiv-1 env and their use |
JP6602304B2 (en) | 2014-01-03 | 2019-11-06 | エフ.ホフマン−ラ ロシュ アーゲー | Covalently linked helicer-anti-helicer antibody conjugates and uses thereof |
BR112016013849A2 (en) | 2014-01-03 | 2017-10-10 | Hoffmann La Roche | bispecific antihapten / blood-brain barrier receptor conjugates, their uses, and pharmaceutical formulation |
CN111057151B (en) | 2014-01-06 | 2022-05-03 | 豪夫迈·罗氏有限公司 | Monovalent blood brain barrier shuttle modules |
MX2016008539A (en) | 2014-01-15 | 2016-09-26 | Hoffmann La Roche | Fc-region variants with modified fcrn- and maintained protein a-binding properties. |
US10150818B2 (en) | 2014-01-16 | 2018-12-11 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
WO2016114819A1 (en) | 2015-01-16 | 2016-07-21 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
KR20160104727A (en) | 2014-01-16 | 2016-09-05 | 아카데미아 시니카 | Compositions and methods for treatment and detection of cancers |
US10179911B2 (en) | 2014-01-20 | 2019-01-15 | President And Fellows Of Harvard College | Negative selection and stringency modulation in continuous evolution systems |
UA119863C2 (en) | 2014-01-24 | 2019-08-27 | Нгм Біофармасьютікалс, Інк. | Binding proteins and methods of use thereof |
JOP20200094A1 (en) | 2014-01-24 | 2017-06-16 | Dana Farber Cancer Inst Inc | Antibody molecules to pd-1 and uses thereof |
JP2017505305A (en) | 2014-01-24 | 2017-02-16 | ジェネンテック, インコーポレイテッド | Methods using anti-STEAP1 antibodies and immunoconjugates |
JOP20200096A1 (en) | 2014-01-31 | 2017-06-16 | Children’S Medical Center Corp | Antibody molecules to tim-3 and uses thereof |
WO2015116902A1 (en) | 2014-01-31 | 2015-08-06 | Genentech, Inc. | G-protein coupled receptors in hedgehog signaling |
US11648335B2 (en) | 2014-01-31 | 2023-05-16 | Wake Forest University Health Sciences | Organ/tissue decellularization, framework maintenance and recellularization |
JP6736467B2 (en) | 2014-02-04 | 2020-08-05 | ジェネンテック, インコーポレイテッド | Smoothing mutant and method of using the same |
WO2015120187A1 (en) | 2014-02-05 | 2015-08-13 | The University Of Chicago | Chimeric antigen receptors recognizing cancer-spevific tn glycopeptide variants |
AU2015214058B2 (en) | 2014-02-08 | 2020-07-09 | Genentech, Inc. | Methods of treating Alzheimer's Disease |
PL3102230T3 (en) | 2014-02-08 | 2021-11-15 | F.Hoffmann-La Roche Ag | Methods of treating alzheimer's disease |
CN106211773B (en) | 2014-02-11 | 2021-09-03 | 威特拉公司 | Antibody molecules for dengue virus and uses thereof |
MX2016010433A (en) | 2014-02-12 | 2016-09-22 | Genentech Inc | Anti-jagged1 antibodies and methods of use. |
US10308721B2 (en) | 2014-02-21 | 2019-06-04 | Abbvie Stemcentrx Llc | Anti-DLL3 antibodies and drug conjugates for use in melanoma |
UA117608C2 (en) | 2014-02-21 | 2018-08-27 | Дженентек, Інк. | Anti-il-13/il-17 bispecific antibodies and uses thereof |
GB201403775D0 (en) | 2014-03-04 | 2014-04-16 | Kymab Ltd | Antibodies, uses & methods |
CN106103484B (en) | 2014-03-14 | 2021-08-20 | 诺华股份有限公司 | Antibody molecules against LAG-3 and uses thereof |
US10435694B2 (en) | 2014-03-14 | 2019-10-08 | Genentech, Inc. | Methods and compositions for secretion of heterologous polypeptides |
US9738702B2 (en) | 2014-03-14 | 2017-08-22 | Janssen Biotech, Inc. | Antibodies with improved half-life in ferrets |
CN106456759A (en) | 2014-03-21 | 2017-02-22 | 泰华制药国际有限公司 | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
US20170107294A1 (en) | 2014-03-21 | 2017-04-20 | Nordlandssykehuset Hf | Anti-cd14 antibodies and uses thereof |
US10556945B2 (en) | 2014-03-21 | 2020-02-11 | Teva Pharmaceuticals International Gmbh | Antagonist antibodies directed against calcitonin gene-related peptide and methods using same |
WO2015148515A1 (en) | 2014-03-24 | 2015-10-01 | Biogen Ma Inc. | Methods for overcoming glutamine deprivation during mammalian cell culture |
JP2017516458A (en) | 2014-03-24 | 2017-06-22 | ジェネンテック, インコーポレイテッド | Cancer treatment with c-met antagonist and correlation with HGF expression of c-met antagonist |
EP3129767B1 (en) | 2014-03-27 | 2021-09-01 | Academia Sinica | Reactive labelling compounds and uses thereof |
MX2016012779A (en) | 2014-03-31 | 2017-04-27 | Genentech Inc | Combination therapy comprising anti-angiogenesis agents and ox40 binding agonists. |
PT3126394T (en) | 2014-03-31 | 2019-12-19 | Hoffmann La Roche | Anti-ox40 antibodies and methods of use |
EP3126389A1 (en) | 2014-04-02 | 2017-02-08 | F. Hoffmann-La Roche AG | Method for detecting multispecific antibody light chain mispairing |
MX2016012873A (en) | 2014-04-04 | 2017-03-07 | Bionomics Inc | Humanized antibodies that bind lgr5. |
CA3208721A1 (en) | 2014-04-08 | 2015-10-15 | Boston Pharmaceuticals Inc. | Binding molecules specific for il-21 and uses thereof |
EP3129055B1 (en) | 2014-04-11 | 2020-07-01 | MedImmune, LLC | Bispecific her2 antibodies |
BR112016024319B1 (en) | 2014-04-18 | 2024-01-23 | Acceleron Pharma Inc | USE OF A COMPOSITION COMPRISING AN ActRII ANTAGONIST FOR THE MANUFACTURING OF A MEDICATION FOR TREATING OR PREVENTING A COMPLICATION OF SICKLE CELL ANEMIA |
WO2015164615A1 (en) | 2014-04-24 | 2015-10-29 | University Of Oslo | Anti-gluten antibodies and uses thereof |
US11427647B2 (en) | 2014-04-27 | 2022-08-30 | Famewave Ltd. | Polynucleotides encoding humanized antibodies against CEACAM1 |
EP3137502B1 (en) | 2014-04-27 | 2020-07-29 | FameWave Ltd. | Humanized antibodies against ceacam1 |
US9753036B2 (en) | 2014-04-29 | 2017-09-05 | Edp Biotech Corporation | Methods and compositions for screening and detecting biomarkers |
AU2015253422B2 (en) | 2014-04-30 | 2020-09-03 | Abbvie Stemcentrx Llc | Anti-PTK7 antibody-drug conjugates |
WO2015171523A1 (en) | 2014-05-05 | 2015-11-12 | Regeneron Pharmaceuticals, Inc. | Humanized c5 and c3 animals |
IL248511B (en) | 2014-05-13 | 2022-07-01 | Bavarian Nordic As | Combination therapy for treating cancer with a poxvirus expressing a tumor antigen and a monoclonal antibody against tim-3 |
EP3888690A3 (en) | 2014-05-16 | 2021-10-20 | MedImmune, LLC | Molecules with altered neonate fc receptor binding having enhanced therapeutic and diagnostic properties |
BR112016027222A2 (en) | 2014-05-22 | 2018-01-30 | Genentech Inc | isolated antibodies, isolated nucleic acid, host cell, method of producing an antibody, immunoconjugate, pharmaceutical formulation, methods of treating an individual with cancer, inhibiting cell proliferation, detecting human gpc3 and detecting a cancer |
KR20170005016A (en) | 2014-05-23 | 2017-01-11 | 제넨테크, 인크. | Mit biomarkers and methods using the same |
US10118969B2 (en) | 2014-05-27 | 2018-11-06 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
EP3149037A4 (en) | 2014-05-27 | 2018-01-10 | Academia Sinica | Anti-her2 glycoantibodies and uses thereof |
EP3149036A4 (en) | 2014-05-27 | 2017-12-27 | Academia Sinica | Anti-cd20 glycoantibodies and uses thereof |
KR20170005142A (en) | 2014-05-27 | 2017-01-11 | 아카데미아 시니카 | Compositions and methods relating to universal glycoforms for enhanced antibody efficacy |
PT3148579T (en) | 2014-05-28 | 2021-03-11 | Ludwig Inst For Cancer Res Ltd | Anti-gitr antibodies and methods of use thereof |
JP7063538B2 (en) | 2014-05-28 | 2022-05-09 | アカデミア シニカ | Anti-TNFα sugar-manipulated antibody group and its use |
GB201409558D0 (en) | 2014-05-29 | 2014-07-16 | Ucb Biopharma Sprl | Method |
EP3970747A3 (en) | 2014-06-03 | 2022-10-05 | XBiotech Inc. | Compositions and methods for treating and preventing staphylococcus aureus infections |
AU2015271685B2 (en) | 2014-06-04 | 2021-02-18 | Biontech Research And Development, Inc. | Human monoclonal antibodies to ganglioside GD2 |
KR20170010785A (en) | 2014-06-11 | 2017-02-01 | 제넨테크, 인크. | Anti-lgr5 antibodies and uses thereof |
BR122023023170A2 (en) | 2014-06-13 | 2024-02-20 | Acceleron Pharma Inc. | USE OF AN ACTRII ANTAGONIST IN THE TREATMENT OR PREVENTION OF SKIN ULCERS ASSOCIATED WITH BETA-THALASSEMIA |
EP3154579A1 (en) | 2014-06-13 | 2017-04-19 | Friedrich Miescher Institute for Biomedical Research | New treatment against influenza virus |
CN107073121A (en) | 2014-06-13 | 2017-08-18 | 基因泰克公司 | Treatment and the method for prevention cancer drug resistance |
US9950077B2 (en) | 2014-06-20 | 2018-04-24 | Bioalliance C.V. | Anti-folate receptor alpha (FRA) antibody-drug conjugates and methods of using thereof |
WO2015198202A1 (en) | 2014-06-23 | 2015-12-30 | Friedrich Miescher Institute For Biomedical Research | Methods for triggering de novo formation of heterochromatin and or epigenetic silencing with small rnas |
GB201411320D0 (en) | 2014-06-25 | 2014-08-06 | Ucb Biopharma Sprl | Antibody construct |
TW201623329A (en) | 2014-06-30 | 2016-07-01 | 亞佛瑞司股份有限公司 | Vaccines and monoclonal antibodies targeting truncated variants of osteopontin and uses thereof |
EP3164129A1 (en) | 2014-07-01 | 2017-05-10 | Friedrich Miescher Institute for Biomedical Research | Combination of a brafv600e inhibitor and mertk inhibitor to treat melanoma |
CA2954687A1 (en) | 2014-07-10 | 2016-01-14 | Affiris Ag | Substances and methods for the use in prevention and/or treatment in huntington's disease |
CN106488775A (en) | 2014-07-11 | 2017-03-08 | 基因泰克公司 | NOTCH approach suppresses |
CN106460067A (en) | 2014-07-14 | 2017-02-22 | 豪夫迈·罗氏有限公司 | Diagnostic methods and compositions for treatment of glioblastoma |
US9139648B1 (en) | 2014-07-15 | 2015-09-22 | Kymab Limited | Precision medicine by targeting human NAV1.9 variants for treatment of pain |
MX2017000546A (en) | 2014-07-15 | 2017-03-08 | Genentech Inc | Compositions for treating cancer using pd-1 axis binding antagonists and mek inhibitors. |
GB201412658D0 (en) | 2014-07-16 | 2014-08-27 | Ucb Biopharma Sprl | Molecules |
GB201412659D0 (en) | 2014-07-16 | 2014-08-27 | Ucb Biopharma Sprl | Molecules |
MY178347A (en) | 2014-07-17 | 2020-10-08 | Novo Nordisk As | Site directed mutagenesis of trem-1 antibodies for decreasing viscosity |
JP6831777B2 (en) | 2014-07-21 | 2021-02-17 | ノバルティス アーゲー | Treatment of cancer using the CD33 chimeric antigen receptor |
JP6940406B2 (en) | 2014-07-25 | 2021-09-29 | テラベクティTheravectys | Lentiviral vector for controlled expression of chimeric antigen receptor molecule |
EP3193928A1 (en) | 2014-08-06 | 2017-07-26 | Rinat Neuroscience Corp. | Methods for reducing ldl-cholesterol |
WO2016020799A1 (en) | 2014-08-06 | 2016-02-11 | Rinat Neuroscience Corp. | Methods for reducing ldl-cholesterol |
US10851149B2 (en) | 2014-08-14 | 2020-12-01 | The Trustees Of The University Of Pennsylvania | Treatment of cancer using GFR α-4 chimeric antigen receptor |
CN107108744B (en) | 2014-08-19 | 2020-09-25 | 诺华股份有限公司 | anti-CD 123 Chimeric Antigen Receptor (CAR) for cancer therapy |
WO2016037157A2 (en) | 2014-09-05 | 2016-03-10 | The Johns Hopkins University | Targeting capn9/capns2 activity as a therapeutic strategy for the treatment of myofibroblast differentiation and associated pathologies |
CA2996445A1 (en) | 2014-09-05 | 2016-03-10 | Eli Hatchwell | Methods and compositions for inhibiting and treating neurological conditions |
TWI745275B (en) | 2014-09-08 | 2021-11-11 | 中央研究院 | HUMAN iNKT CELL ACTIVATION USING GLYCOLIPIDS |
CN113698488A (en) | 2014-09-12 | 2021-11-26 | 基因泰克公司 | anti-B7-H4 antibodies and immunoconjugates |
CA2958479A1 (en) | 2014-09-12 | 2016-03-17 | Genentech, Inc. | Anti-cll-1 antibodies and immunoconjugates |
SI3191135T1 (en) | 2014-09-12 | 2021-01-29 | Genentech, Inc. | Anti-her2 antibodies and immunoconjugates |
CN107206071A (en) | 2014-09-13 | 2017-09-26 | 诺华股份有限公司 | The conjoint therapy of ALK inhibitor |
CN106794247B (en) | 2014-09-15 | 2022-12-02 | 豪夫迈·罗氏有限公司 | Antibody formulations |
BR112017005202A2 (en) | 2014-09-16 | 2017-12-12 | Symphogen As | anti-met antibodies and compositions |
BR112017004953A2 (en) | 2014-09-17 | 2017-12-05 | Genentech Inc | immunoconjugate, pharmaceutical formulation, method of treatment and method of inhibiting cell proliferation |
WO2016044697A1 (en) | 2014-09-19 | 2016-03-24 | The Johns Hopkins University | Biomarkers of cognitive dysfunction |
CA3069221C (en) | 2014-09-23 | 2023-04-04 | Genentech, Inc. | Methods of using anti-cd79b immunoconjugates |
EP3197492A1 (en) | 2014-09-23 | 2017-08-02 | Pfizer Inc | Treatment with anti-pcsk9 antibodies |
WO2016046768A1 (en) | 2014-09-24 | 2016-03-31 | Friedrich Miescher Institute For Biomedical Research | Lats and breast cancer |
RU2754684C2 (en) | 2014-09-30 | 2021-09-06 | Дойчес Кребсфоршунгсцентрум Штифтунг Дес Эффентлихен Рехтс | Binding molecules, namely antibodies capable of binding to l1cam (cd171) |
CN113956361A (en) | 2014-10-01 | 2022-01-21 | 免疫医疗有限公司 | Antibodies to ticagrelor and methods of use thereof |
US20170209574A1 (en) | 2014-10-03 | 2017-07-27 | Novartis Ag | Combination therapies |
UY36351A (en) | 2014-10-14 | 2016-06-01 | Novartis Ag | ANTIBODY MOLECULES THAT JOIN PD-L1 AND USES OF THE SAME |
JP2017536102A (en) | 2014-10-16 | 2017-12-07 | ジェネンテック, インコーポレイテッド | Anti-alpha-synuclein antibodies and methods of use |
US11124822B2 (en) | 2014-10-17 | 2021-09-21 | Carnegie Mellon University | Enhanced biomolecule detection assays based on tyramide signal amplification and gammaPNA probes |
MA41685A (en) | 2014-10-17 | 2017-08-22 | Biogen Ma Inc | COPPER SUPPLEMENT FOR THE REGULATION OF GLYCOSYLATION IN A MAMMAL CELL CULTURE PROCESS |
US10920208B2 (en) | 2014-10-22 | 2021-02-16 | President And Fellows Of Harvard College | Evolution of proteases |
WO2016061632A1 (en) | 2014-10-23 | 2016-04-28 | La Trobe University | Fn14-binding proteins and uses thereof |
LT3412302T (en) | 2014-10-24 | 2021-07-26 | Bristol-Myers Squibb Company | Modified fgf-21 polypeptides and uses thereof |
EP3223865A4 (en) | 2014-10-31 | 2018-10-03 | Jounce Therapeutics, Inc. | Methods of treating conditions with antibodies that bind b7-h4 |
AU2015338974B2 (en) | 2014-10-31 | 2021-08-26 | Oncomed Pharmaceuticals, Inc. | Combination therapy for treatment of disease |
MA40864A (en) | 2014-10-31 | 2017-09-05 | Biogen Ma Inc | HYPOTAURINE, GABA, BETA-ALANINE AND CHOLINE FOR THE REGULATION OF THE ACCUMULATION OF RESIDUAL BY-PRODUCTS IN MAMMAL CELL CULTURE PROCESSES |
US20160160290A1 (en) | 2014-11-03 | 2016-06-09 | Genentech, Inc. | Methods and biomarkers for predicting efficacy and evaluation of an ox40 agonist treatment |
EP3215850B1 (en) | 2014-11-03 | 2019-07-03 | F. Hoffmann-La Roche AG | Assays for detecting t cell immune subsets and methods of use thereof |
JP6875276B2 (en) | 2014-11-05 | 2021-05-19 | ジェネンテック, インコーポレイテッド | Method of producing double-stranded protein in bacteria |
RU2017119185A (en) | 2014-11-05 | 2018-12-05 | Дженентек, Инк. | ANTIBODIES AGAINST FGFR2 / 3 AND WAYS OF THEIR APPLICATION |
EP3215527A4 (en) | 2014-11-05 | 2018-04-18 | Annexon, Inc. | Humanized anti-complement factor c1q antibodies and uses thereof |
RU2020141422A (en) | 2014-11-05 | 2021-01-13 | Дженентек, Инк. | METHODS FOR OBTAINING TWO-STRAIN PROTEINS IN BACTERIA |
WO2016073157A1 (en) | 2014-11-06 | 2016-05-12 | Genentech, Inc. | Anti-ang2 antibodies and methods of use thereof |
PL3215528T3 (en) | 2014-11-06 | 2020-01-31 | F.Hoffmann-La Roche Ag | Fc-region variants with modified fcrn-binding and methods of use |
CN107074966A (en) | 2014-11-06 | 2017-08-18 | 豪夫迈·罗氏有限公司 | The Fc region variants of FCRN and albumin A binding property with change |
US20160152720A1 (en) | 2014-11-06 | 2016-06-02 | Genentech, Inc. | Combination therapy comprising ox40 binding agonists and tigit inhibitors |
WO2016073894A1 (en) | 2014-11-07 | 2016-05-12 | Eleven Biotherapeutics, Inc. | Therapeutic agents with increased ocular retention |
KR102636726B1 (en) | 2014-11-07 | 2024-02-13 | 에프. 호프만 라-로셰 엘티디 | Improved il-6 antibodies |
CN107105632A (en) | 2014-11-10 | 2017-08-29 | 豪夫迈·罗氏有限公司 | Nephrosis animal model and its therapeutic agent |
JP6755866B2 (en) | 2014-11-10 | 2020-09-16 | メディミューン リミテッド | CD73-specific binding molecule and its use |
EA201791029A1 (en) | 2014-11-10 | 2017-12-29 | Дженентек, Инк. | ANTIBODIES AGAINST INTERLEUKIN-33 AND THEIR APPLICATION |
GB2538120A (en) | 2014-11-11 | 2016-11-09 | Medimmune Ltd | Therapeutic combinations comprising anti-CD73 antibodies and uses thereof |
US9879087B2 (en) | 2014-11-12 | 2018-01-30 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
WO2016077526A1 (en) | 2014-11-12 | 2016-05-19 | Siamab Therapeutics, Inc. | Glycan-interacting compounds and methods of use |
EP3875481A1 (en) | 2014-11-14 | 2021-09-08 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Neutralizing antibodies to ebola virus glycoprotein and their use |
AU2015350242A1 (en) | 2014-11-17 | 2017-06-29 | Genentech, Inc. | Combination therapy comprising OX40 binding agonists and PD-1 axis binding antagonists |
US10434177B2 (en) | 2014-11-17 | 2019-10-08 | Carnegie Mellon University | Activatable two-component photosensitizers |
EP3221361B1 (en) | 2014-11-19 | 2021-04-21 | Genentech, Inc. | Anti-transferrin receptor / anti-bace1 multispecific antibodies and methods of use |
EP3221362B1 (en) | 2014-11-19 | 2019-07-24 | F.Hoffmann-La Roche Ag | Anti-transferrin receptor antibodies and methods of use |
JP6859259B2 (en) | 2014-11-19 | 2021-04-14 | ジェネンテック, インコーポレイテッド | Antibodies to BACEl and its use for neurological disease immunotherapy |
CA2966566C (en) | 2014-11-20 | 2024-03-19 | F. Hoffmann-La Roche Ag | Combination therapy of t cell activating bispecific antigen binding molecules cd3 and folate receptor 1 (folr1) and pd-1 axis binding antagonists |
ES2832802T3 (en) | 2014-11-21 | 2021-06-11 | Univ Maryland | Systems of directed administration of the specific particulate of a structure |
MA41119A (en) | 2014-12-03 | 2017-10-10 | Acceleron Pharma Inc | METHODS OF TREATMENT OF MYELODYSPLASIC SYNDROMES AND SIDEROBLASTIC ANEMIA |
HUE045216T2 (en) | 2014-12-05 | 2019-12-30 | Hoffmann La Roche | Anti-cd79b antibodies and methods of use |
US20160158360A1 (en) | 2014-12-05 | 2016-06-09 | Genentech, Inc. | Methods and compositions for treating cancer using pd-1 axis antagonists and hpk1 antagonists |
AU2015360903B2 (en) | 2014-12-08 | 2021-03-25 | Dana-Farber Cancer Institute, Inc. | Methods for upregulating immune responses using combinations of anti-RGMB and anti-PD-1 agents |
SG11201703750XA (en) | 2014-12-10 | 2017-06-29 | Genentech Inc | Blood brain barrier receptor antibodies and methods of use |
WO2016094881A2 (en) | 2014-12-11 | 2016-06-16 | Abbvie Inc. | Lrp-8 binding proteins |
EP3333191B1 (en) | 2014-12-11 | 2020-09-09 | Pierre Fabre Medicament | Anti-c10orf54 antibodies and uses thereof |
US20170340733A1 (en) | 2014-12-19 | 2017-11-30 | Novartis Ag | Combination therapies |
EA201791366A1 (en) | 2014-12-19 | 2018-02-28 | Чугаи Сейяку Кабусики Кайся | ANTIBODIES TO C5 AND METHODS OF THEIR APPLICATION |
WO2016106221A1 (en) | 2014-12-22 | 2016-06-30 | The Rockefeller University | Anti-mertk agonistic antibodies and uses thereof |
WO2016106286A1 (en) | 2014-12-23 | 2016-06-30 | Biodesy, Inc. | Attachment of proteins to interfaces for use in nonlinear optical detection |
US20160200815A1 (en) | 2015-01-05 | 2016-07-14 | Jounce Therapeutics, Inc. | Antibodies that inhibit tim-3:lilrb2 interactions and uses thereof |
JP2018504400A (en) | 2015-01-08 | 2018-02-15 | バイオジェン・エムエイ・インコーポレイテッドBiogen MA Inc. | LINGO-1 antagonist and use for treatment of demyelinating disorders |
EP3242685B1 (en) | 2015-01-09 | 2022-05-04 | AdAlta Limited | Cxcr4 binding molecules |
US10495645B2 (en) | 2015-01-16 | 2019-12-03 | Academia Sinica | Cancer markers and methods of use thereof |
US9975965B2 (en) | 2015-01-16 | 2018-05-22 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
WO2016117346A1 (en) | 2015-01-22 | 2016-07-28 | Chugai Seiyaku Kabushiki Kaisha | A combination of two or more anti-c5 antibodies and methods of use |
WO2016118961A1 (en) | 2015-01-24 | 2016-07-28 | Academia Sinica | Cancer markers and methods of use thereof |
AU2015378564A1 (en) | 2015-01-24 | 2017-07-13 | Academia Sinica | Novel glycan conjugates and methods of use thereof |
JP7264592B2 (en) | 2015-01-26 | 2023-04-25 | ザ ユニバーシティー オブ シカゴ | IL13Rα2 BINDING AGENTS AND THEIR USE IN CANCER THERAPY |
US20180170992A1 (en) | 2015-01-26 | 2018-06-21 | The University Of Chicago | CAR T CELLS RECOGNIZING CANCER-SPECIFIC IL 13Ra2 |
EP3250590B1 (en) | 2015-01-30 | 2021-09-15 | Academia Sinica | Compositions and methods relating to universal glycoforms for enhanced anti-ssea4 antibody efficacy |
CA3185253A1 (en) | 2015-02-02 | 2016-08-11 | Children's Health Care D/B/A Children's Minnesota | Anti-surrogate light chain antibodies |
EP3653712B1 (en) | 2015-02-04 | 2022-11-30 | F. Hoffmann-La Roche AG | Tau antisense oligomers and uses thereof |
CA2975875A1 (en) | 2015-02-04 | 2016-08-11 | Genentech, Inc. | Mutant smoothened and methods of using the same |
CN107636159B (en) | 2015-02-04 | 2022-06-14 | 百时美施贵宝公司 | Method for selecting therapeutic molecules |
CN107108729A (en) | 2015-02-05 | 2017-08-29 | 中外制药株式会社 | The antibody of antigen-binding domains comprising ion concentration dependence, FC region variants, the binding antibodies of IL 8, and its application |
EP3265491A1 (en) | 2015-03-03 | 2018-01-10 | Xoma (Us) Llc | Treatment of post-prandial hyperinsulinemia and hypoglycemia after bariatric surgery |
CN114504651A (en) | 2015-03-03 | 2022-05-17 | 科马布有限公司 | Antibodies, uses and methods |
US20180042998A1 (en) | 2015-03-10 | 2018-02-15 | University Of Massachusetts | Targeting gdf6 and bmp signaling for anti-melanoma therapy |
WO2016149276A1 (en) | 2015-03-16 | 2016-09-22 | Genentech, Inc. | Methods of detecting and quantifying il-13 and uses in diagnosing and treating th2-associated diseases |
WO2016146833A1 (en) | 2015-03-19 | 2016-09-22 | F. Hoffmann-La Roche Ag | Biomarkers for nad(+)-diphthamide adp ribosyltransferase resistance |
US10174292B2 (en) | 2015-03-20 | 2019-01-08 | International Aids Vaccine Initiative | Soluble HIV-1 envelope glycoprotein trimers |
US10562960B2 (en) | 2015-03-20 | 2020-02-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to gp120 and their use |
PT3273992T (en) | 2015-03-23 | 2020-08-21 | Jounce Therapeutics Inc | Antibodies to icos |
US9931394B2 (en) | 2015-03-23 | 2018-04-03 | International Aids Vaccine Initiative | Soluble HIV-1 envelope glycoprotein trimers |
US10376535B2 (en) | 2015-03-26 | 2019-08-13 | University Of Rochester | Therapy for malignant disease |
JP2018518152A (en) | 2015-03-27 | 2018-07-12 | ユニバーシティ オブ サザン カリフォルニア | CAR T cell therapy directed to LHR for treating solid tumors |
CN108136001B (en) | 2015-04-03 | 2022-07-29 | 佐马技术有限公司 | Treatment of cancer using TGF-beta inhibitors and PD-1 inhibitors |
KR20170132793A (en) | 2015-04-03 | 2017-12-04 | 유레카 쎄라퓨틱스, 인코포레이티드 | Constructs targeting AFP peptide / MHC complexes and uses thereof |
BR112017021536A2 (en) | 2015-04-06 | 2018-07-03 | Harvard College | compositions and methods for nonmyeloablative conditioning |
MA41919A (en) | 2015-04-06 | 2018-02-13 | Acceleron Pharma Inc | ALK4 HETEROMULTIMERS: ACTRIIB AND THEIR USES |
MA53400A (en) | 2015-04-06 | 2021-08-04 | Acceleron Pharma Inc | ALK7 HETEROMULTIMERS: ACTRIIB AND THEIR USES |
EP3280441B1 (en) | 2015-04-07 | 2021-09-29 | Alector LLC | Anti-sortilin antibodies and methods of use thereof |
WO2016164480A1 (en) | 2015-04-07 | 2016-10-13 | Genentech, Inc. | Antigen binding complex having agonistic activity and methods of use |
JP6921001B2 (en) | 2015-04-13 | 2021-08-18 | ファイザー・インク | Chimeric antigen receptor that targets B cell maturation antigens |
TWI703159B (en) | 2015-04-13 | 2020-09-01 | 美商輝瑞股份有限公司 | Bcma-specific therapeutic antibodies and their uses |
US11299729B2 (en) | 2015-04-17 | 2022-04-12 | President And Fellows Of Harvard College | Vector-based mutagenesis system |
JP2018512856A (en) | 2015-04-17 | 2018-05-24 | アルパイン イミューン サイエンシズ インコーポレイテッド | Immunomodulating proteins with tunable affinity |
WO2016170022A1 (en) | 2015-04-21 | 2016-10-27 | Institut Gustave Roussy | Therapeutic methods, products and compositions inhibiting znf555 |
GB201506869D0 (en) | 2015-04-22 | 2015-06-03 | Ucb Biopharma Sprl | Method |
GB201506870D0 (en) | 2015-04-22 | 2015-06-03 | Ucb Biopharma Sprl | Method |
CN107810197B (en) | 2015-04-24 | 2022-10-25 | 豪夫迈·罗氏有限公司 | Methods of identifying bacteria comprising binding polypeptides |
EP3288542B1 (en) | 2015-04-29 | 2021-12-29 | University Of South Australia | Compositions and methods for administering antibodies |
EP3288981A1 (en) | 2015-05-01 | 2018-03-07 | Genentech, Inc. | Masked anti-cd3 antibodies and methods of use |
WO2016179194A1 (en) | 2015-05-04 | 2016-11-10 | Jounce Therapeutics, Inc. | Lilra3 and method of using the same |
AU2016258115A1 (en) | 2015-05-06 | 2017-11-23 | Janssen Biotech, Inc. | Prostate specific membrane antigen (PSMA) bispecific binding agents and uses thereof |
MA42043A (en) | 2015-05-07 | 2018-03-14 | Agenus Inc | ANTI-OX40 ANTIBODIES AND METHODS OF USE THEREOF |
US20180104331A1 (en) | 2015-05-11 | 2018-04-19 | The Johns Hopkins University | Autoimmune antibodies for use in inhibiting cancer cell growth |
CN107592812A (en) | 2015-05-11 | 2018-01-16 | 豪夫迈·罗氏有限公司 | Treat the composition and method of lupus nephritis |
IL255312B (en) | 2015-05-12 | 2022-08-01 | Genentech Inc | Therapeutic and diagnostic methods for cancer comprising a pd-l1 binding antagonist |
AU2016267059B2 (en) | 2015-05-22 | 2020-08-13 | Translational Drug Development Llc | Benzamide and active compound compositions and methods of use |
CN113940996A (en) | 2015-05-27 | 2022-01-18 | Ucb生物制药私人有限公司 | Methods for treating neurological diseases |
US10174121B2 (en) | 2015-05-29 | 2019-01-08 | Abbvie, Inc. | Anti-CD40 antibodies |
EP3303394B1 (en) | 2015-05-29 | 2020-04-08 | Agenus Inc. | Anti-ctla-4 antibodies and methods of use thereof |
JP2018520658A (en) | 2015-05-29 | 2018-08-02 | ジェネンテック, インコーポレイテッド | Humanized anti-Ebola virus glycoprotein antibodies and uses thereof |
EP3708681A1 (en) | 2015-05-29 | 2020-09-16 | F. Hoffmann-La Roche AG | Therapeutic and diagnostic methods for cancer |
CA2984638A1 (en) | 2015-05-29 | 2016-12-08 | Genentech, Inc. | Pd-l1 promoter methylation in cancer |
EP3302552A1 (en) | 2015-06-02 | 2018-04-11 | H. Hoffnabb-La Roche Ag | Compositions and methods for using anti-il-34 antibodies to treat neurological diseases |
US11208452B2 (en) | 2015-06-02 | 2021-12-28 | Novo Nordisk A/S | Insulins with polar recombinant extensions |
WO2016196975A1 (en) | 2015-06-03 | 2016-12-08 | The United States Of America, As Represented By The Secretary Department Of Health & Human Services | Neutralizing antibodies to hiv-1 env and their use |
EP3302559B1 (en) | 2015-06-04 | 2022-01-12 | University of Southern California | Lym-1 and lym-2 targeted car cell immunotherapy |
PE20180317A1 (en) | 2015-06-05 | 2018-02-09 | Genentech Inc | ANTI-TAU ANTIBODIES AND METHODS OF USE |
EP3303397A1 (en) | 2015-06-08 | 2018-04-11 | H. Hoffnabb-La Roche Ag | Methods of treating cancer using anti-ox40 antibodies and pd-1 axis binding antagonists |
CN107810011A (en) | 2015-06-08 | 2018-03-16 | 豪夫迈·罗氏有限公司 | Use the method for anti-OX40 antibodies for treating cancer |
US11174313B2 (en) | 2015-06-12 | 2021-11-16 | Alector Llc | Anti-CD33 antibodies and methods of use thereof |
CA2988982A1 (en) | 2015-06-12 | 2016-12-15 | Alector Llc | Anti-cd33 antibodies and methods of use thereof |
TW201709934A (en) | 2015-06-15 | 2017-03-16 | 建南德克公司 | Antibodies and immunoconjugates |
TW201710286A (en) | 2015-06-15 | 2017-03-16 | 艾伯維有限公司 | Binding proteins against VEGF, PDGF, and/or their receptors |
US10501545B2 (en) | 2015-06-16 | 2019-12-10 | Genentech, Inc. | Anti-CLL-1 antibodies and methods of use |
IL256079B2 (en) | 2015-06-16 | 2024-01-01 | Genentech Inc | Humanized and affinity matured antibodies to fcrh5 and methods of use |
CN107849145B (en) | 2015-06-16 | 2021-10-26 | 基因泰克公司 | anti-CD 3 antibodies and methods of use thereof |
CA2986263A1 (en) | 2015-06-17 | 2016-12-22 | Genentech, Inc. | Methods of treating locally advanced or metastatic breast cancers using pd-1 axis binding antagonists and taxanes |
KR20180012859A (en) | 2015-06-17 | 2018-02-06 | 제넨테크, 인크. | Anti-HER2 antibodies and methods of use |
GB201510758D0 (en) | 2015-06-18 | 2015-08-05 | Ucb Biopharma Sprl | Novel TNFa structure for use in therapy |
EP3311165B1 (en) | 2015-06-19 | 2020-12-09 | University Of Rochester | Septin proteins as novel biomarkers for detection and treatment of müllerian cancers |
CN107922497B (en) | 2015-06-24 | 2022-04-12 | 詹森药业有限公司 | anti-VISTA antibodies and fragments |
PL3313884T3 (en) | 2015-06-29 | 2021-05-31 | Immunogen, Inc. | Anti-cd123 antibodies and conjugates and derivatives thereof |
MX2017016645A (en) | 2015-06-29 | 2018-11-09 | Genentech Inc | Type ii anti-cd20 antibody for use in organ transplantation. |
GB201601075D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibodies molecules |
GB201601073D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibodies |
GB201601077D0 (en) | 2016-01-20 | 2016-03-02 | Ucb Biopharma Sprl | Antibody molecule |
US10877045B2 (en) | 2015-07-21 | 2020-12-29 | Saint Louis University | Compositions and methods for diagnosing and treating endometriosis-related infertility |
WO2017015545A1 (en) | 2015-07-22 | 2017-01-26 | President And Fellows Of Harvard College | Evolution of site-specific recombinases |
WO2017015559A2 (en) | 2015-07-23 | 2017-01-26 | President And Fellows Of Harvard College | Evolution of bt toxins |
US20180207273A1 (en) | 2015-07-29 | 2018-07-26 | Novartis Ag | Combination therapies comprising antibody molecules to tim-3 |
EP3964528A1 (en) | 2015-07-29 | 2022-03-09 | Novartis AG | Combination therapies comprising antibody molecules to lag-3 |
EP4378957A2 (en) | 2015-07-29 | 2024-06-05 | Novartis AG | Combination therapies comprising antibody molecules to pd-1 |
US10612011B2 (en) | 2015-07-30 | 2020-04-07 | President And Fellows Of Harvard College | Evolution of TALENs |
US9884900B2 (en) | 2015-08-04 | 2018-02-06 | Acceleron Pharma Inc. | Methods for treating Janus kinase-associated disorders by administering soluble transforming growth factor beta type II receptor |
TW202330904A (en) | 2015-08-04 | 2023-08-01 | 美商再生元醫藥公司 | Taurine supplemented cell culture medium and methods of use |
JP2018532693A (en) | 2015-08-06 | 2018-11-08 | ゾーマ (ユーエス) リミテッド ライアビリティ カンパニー | Antibody fragments against the insulin receptor and their use to treat hypoglycemia |
CN105384825B (en) | 2015-08-11 | 2018-06-01 | 南京传奇生物科技有限公司 | A kind of bispecific chimeric antigen receptor and its application based on single domain antibody |
EP3341415B1 (en) | 2015-08-28 | 2021-03-24 | H. Hoffnabb-La Roche Ag | Anti-hypusine antibodies and uses thereof |
EA201890630A1 (en) | 2015-09-01 | 2018-10-31 | Эйдженус Инк. | ANTIBODIES AGAINST PD-1 AND METHODS OF THEIR APPLICATION |
CN108350605A (en) | 2015-09-04 | 2018-07-31 | 台湾浩鼎生技股份有限公司 | Glycan array and application method |
IL258121B2 (en) | 2015-09-15 | 2024-01-01 | Scholar Rock Inc | Anti-pro/latent-myostatin antibodies and uses thereof |
AU2016323968B2 (en) | 2015-09-17 | 2023-07-06 | Immunogen, Inc. | Therapeutic combinations comprising anti-FOLR1 immunoconjugates |
BR112018002432A2 (en) | 2015-09-18 | 2018-09-18 | Chugai Pharmaceutical Co Ltd | il-8 binding antibodies and uses thereof |
JP7054924B2 (en) | 2015-09-23 | 2022-04-15 | サイトイミューン セラピューティクス, インコーポレイテッド | FLT3-oriented CAR cells for immunotherapy |
CN109071644B (en) | 2015-09-23 | 2023-09-19 | 昂考梅德药品有限公司 | Methods and compositions for treating cancer |
BR112018005737A2 (en) | 2015-09-23 | 2018-10-09 | Genentech Inc | antibodies, polynucleotide, vector, host cell, method for producing antibody, for reducing or inhibiting angiogenesis, for treating a disorder associated with angiogenesis, for inhibiting vascular permeability, composition, antibody conjugate, fusion protein, for identifying a change residues, antibody use, conjugate use and protein use |
KR20180083313A (en) | 2015-09-24 | 2018-07-20 | 에이비비트로, 엘엘씨 | HIV antibody compositions and methods of use |
RU2732591C2 (en) | 2015-09-25 | 2020-09-21 | Дженентек, Инк. | Anti-tigit antibodies and methods of using |
BR112018006237A2 (en) | 2015-09-29 | 2018-10-09 | Celgene Corp | pd-1 binding proteins and methods of using them |
JP2018529719A (en) | 2015-09-30 | 2018-10-11 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung | Combination of PD-1 system binding antagonist and ALK inhibitor for treating ALK negative cancer |
MA43348A (en) | 2015-10-01 | 2018-08-08 | Novo Nordisk As | PROTEIN CONJUGATES |
PE20181092A1 (en) | 2015-10-02 | 2018-07-09 | Hoffmann La Roche | ANTI-PD1 ANTIBODIES AND METHODS OF USE |
MA43345A (en) | 2015-10-02 | 2018-08-08 | Hoffmann La Roche | PYRROLOBENZODIAZEPINE ANTIBODY-DRUG CONJUGATES AND METHODS OF USE |
US11161912B2 (en) | 2015-10-13 | 2021-11-02 | Technion Research & Development Foundation Limited | Heparanase-neutralizing monoclonal antibodies |
EP3362074B1 (en) | 2015-10-16 | 2023-08-09 | President and Fellows of Harvard College | Regulatory t cell pd-1 modulation for regulating t cell effector immune responses |
MA43354A (en) | 2015-10-16 | 2018-08-22 | Genentech Inc | CONJUGATE DRUG CONJUGATES WITH CLOUDY DISULPHIDE |
MA45326A (en) | 2015-10-20 | 2018-08-29 | Genentech Inc | CALICHEAMICIN-ANTIBODY-DRUG CONJUGATES AND METHODS OF USE |
US10968277B2 (en) | 2015-10-22 | 2021-04-06 | Jounce Therapeutics, Inc. | Gene signatures for determining ICOS expression |
CN115927191A (en) | 2015-10-23 | 2023-04-07 | 优瑞科生物技术公司 | Antibody/T cell receptor chimeric constructs and uses thereof |
CN116059350A (en) | 2015-10-27 | 2023-05-05 | Ucb生物制药有限责任公司 | Methods of treatment using anti-IL-17A/F antibodies |
US20180348224A1 (en) | 2015-10-28 | 2018-12-06 | Friedrich Miescher Institute For Biomedical Resear Ch | Tenascin-w and biliary tract cancers |
US20210106661A1 (en) | 2015-10-29 | 2021-04-15 | Dana-Farber Cancer Institute, Inc. | Methods for identification, assessment, prevention, and treatment of metabolic disorders using pm20d1 and n-lipidated amino acids |
EP3184547A1 (en) | 2015-10-29 | 2017-06-28 | F. Hoffmann-La Roche AG | Anti-tpbg antibodies and methods of use |
KR102162324B1 (en) | 2015-10-30 | 2020-10-07 | 제넨테크, 인크. | Anti-HtrA1 antibodies and methods of use thereof |
US10407510B2 (en) | 2015-10-30 | 2019-09-10 | Genentech, Inc. | Anti-factor D antibodies and conjugates |
GB201519303D0 (en) | 2015-11-02 | 2015-12-16 | Imp Innovations Ltd | Phagemid vector |
US11273151B2 (en) | 2015-11-04 | 2022-03-15 | Icahn School Of Medicine At Mount Sinai | Methods of treating tumors and cancer, and identifying candidate subjects for such treatment |
WO2017079768A1 (en) | 2015-11-08 | 2017-05-11 | Genentech, Inc. | Methods of screening for multispecific antibodies |
DK3374398T3 (en) | 2015-11-10 | 2020-06-08 | Medimmune Llc | BINDING MOLECULES SPECIFIC TO ASCT2 AND APPLICATIONS THEREOF |
EP3374391B1 (en) | 2015-11-10 | 2024-04-17 | Visterra, Inc. | Antibody molecule-drug conjugates that specifically binds to lipopolysaccharide and uses thereof |
CN116217729A (en) | 2015-11-12 | 2023-06-06 | 思进公司 | Glycan interaction compounds and methods of use |
CA3005975A1 (en) | 2015-11-23 | 2017-06-01 | Acceleron Pharma Inc. | Methods for treating eye disorders |
CN118027201A (en) | 2015-11-25 | 2024-05-14 | 威特拉公司 | Antibody molecules to APRIL and uses thereof |
KR20180100122A (en) | 2015-12-02 | 2018-09-07 | 주식회사 에스티사이언스 | Antibodies specific for glycated BTLA (B- and T-lymphocyte weakening factor) |
ES2861449T3 (en) | 2015-12-02 | 2021-10-06 | Stcube & Co Inc | Antibodies and Molecules that Immunospecifically Bind to BTN1A1 and Their Therapeutic Uses |
GB201521382D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521393D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521383D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl And Ucb Celltech | Method |
GB201521391D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Antibodies |
GB201521389D0 (en) | 2015-12-03 | 2016-01-20 | Ucb Biopharma Sprl | Method |
EP3178848A1 (en) | 2015-12-09 | 2017-06-14 | F. Hoffmann-La Roche AG | Type ii anti-cd20 antibody for reducing formation of anti-drug antibodies |
CN115920030A (en) | 2015-12-09 | 2023-04-07 | 豪夫迈·罗氏有限公司 | Use of type II anti-CD 20 antibodies for reducing anti-drug antibody formation |
WO2017097889A1 (en) | 2015-12-10 | 2017-06-15 | Katholieke Universiteit Leuven | Anti adamts13 antibodies and their use for treatment or prevention of haemorrhagic disorders due to ventricular assist device |
JP2019503349A (en) | 2015-12-17 | 2019-02-07 | ノバルティス アーゲー | Antibody molecules against PD-1 and uses thereof |
KR20180094977A (en) | 2015-12-17 | 2018-08-24 | 노파르티스 아게 | Combinations of c-Met inhibitors and antibody molecules for PD-1 and uses thereof |
SG10201709415WA (en) | 2015-12-18 | 2017-12-28 | Chugai Pharmaceutical Co Ltd | Anti-c5 antibodies and methods of use |
CA3048202A1 (en) | 2015-12-23 | 2017-06-29 | Moonshot Pharma Llc | Methods for inducing an immune response by inhibition of nonsense mediated decay |
KR20180093078A (en) | 2015-12-30 | 2018-08-20 | 제넨테크, 인크. | Use of tryptophan derivatives for protein preparations |
KR20180098625A (en) | 2015-12-30 | 2018-09-04 | 제넨테크, 인크. | Formulation with reduced degradation of polysorbate |
CN107531795B (en) | 2016-01-05 | 2021-01-19 | 江苏恒瑞医药股份有限公司 | PCSK9 antibody, antigen-binding fragment thereof and medical application thereof |
MX2018008369A (en) | 2016-01-08 | 2019-05-15 | Scholar Rock Inc | Anti-pro/latent myostatin antibodies and methods of use thereof. |
CN108368179B (en) | 2016-01-08 | 2022-08-23 | 豪夫迈·罗氏有限公司 | Methods of treating CEA positive cancers using PD-1 axis binding antagonists and anti-CEA/anti-CD 3 bispecific antibodies |
BR112018013912A2 (en) | 2016-01-08 | 2018-12-18 | Bioalliance C.V. | tetravalent anti-psgl-1 antibodies and uses thereof |
US20190016791A1 (en) | 2016-01-20 | 2019-01-17 | Genentech, Inc. | High dose treatments for alzheimer's disease |
AU2017208834B2 (en) | 2016-01-21 | 2021-09-02 | Pfizer Inc. | Chimeric Antigen Receptors targeting Epidermal Growth Factor Receptor Variant III |
EP3405490B1 (en) | 2016-01-21 | 2021-10-20 | Pfizer Inc. | Mono and bispecific antibodies for epidermal growth factor receptor variant iii and cd3 and their uses |
CN109071625A (en) | 2016-02-04 | 2018-12-21 | 柯瑞斯公司 | Smooth mutant and its application method |
GB201602413D0 (en) | 2016-02-10 | 2016-03-23 | Nascient Ltd | Method |
TWI756204B (en) | 2016-02-12 | 2022-03-01 | 比利時商楊森製藥公司 | Anti-vista antibodies and fragments, uses thereof, and methods of identifying same |
WO2017151517A1 (en) | 2016-02-29 | 2017-09-08 | Foundation Medicine, Inc. | Methods of treating cancer |
AU2017225854B2 (en) | 2016-02-29 | 2020-11-19 | Foundation Medicine, Inc. | Therapeutic and diagnostic methods for cancer |
CA3016170A1 (en) | 2016-03-08 | 2017-09-14 | Academia Sinica | Methods for modular synthesis of n-glycans and arrays thereof |
EP3426680A4 (en) | 2016-03-10 | 2020-03-11 | Acceleron Pharma Inc. | Activin type 2 receptor binding proteins and uses thereof |
KR102632796B1 (en) | 2016-03-10 | 2024-02-02 | 비엘라 바이오, 인크. | Ilt7 binding molecules and methods of using the same |
MX2018010948A (en) | 2016-03-11 | 2019-06-20 | Scholar Rock Inc | TGFß1-BINDING IMMUNOGLOBULINS AND USE THEREOF. |
WO2017160599A1 (en) | 2016-03-14 | 2017-09-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Use of cd300b antagonists to treat sepsis and septic shock |
TWI759287B (en) | 2016-03-15 | 2022-04-01 | 日商中外製藥股份有限公司 | Methods of treating cancers using pd-1 axis binding antagonists and anti-gpc3 antibodies |
WO2017158436A1 (en) | 2016-03-17 | 2017-09-21 | Oslo Universitetssykehus Hf | Fusion proteins targeting tumour associated macrophages for treating cancer |
EP3433281A1 (en) | 2016-03-21 | 2019-01-30 | Elstar Therapeutics, Inc. | Multispecific and multifunctional molecules and uses thereof |
CN108697799A (en) | 2016-03-22 | 2018-10-23 | 生态学有限公司 | The application of anti-LGR5 monoclonal antibodies |
JP6943872B2 (en) | 2016-03-25 | 2021-10-06 | ジェネンテック, インコーポレイテッド | Multiple whole antibody and antibody complex drug quantification assay |
CA3018216A1 (en) | 2016-03-25 | 2017-09-28 | Visterra, Inc. | Formulations of antibody molecules to dengue virus |
US10980894B2 (en) | 2016-03-29 | 2021-04-20 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
AU2017239637A1 (en) | 2016-03-29 | 2018-11-15 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
BR112018069776A2 (en) | 2016-03-29 | 2019-02-05 | Janssen Biotech Inc | psoriasis treatment with increased dosage range of anti-il-12 and / or anti-il-23 antibodies |
WO2017175058A1 (en) | 2016-04-07 | 2017-10-12 | Janssen Pharmaceutica Nv | Anti-vista antibodies and fragments, uses thereof, and methods of identifying same |
JP7197078B2 (en) | 2016-04-08 | 2022-12-27 | ジィールバイオ,インコーポレーテッド | Plectin-1 binding antibody and use thereof |
WO2017180864A1 (en) | 2016-04-14 | 2017-10-19 | Genentech, Inc. | Anti-rspo3 antibodies and methods of use |
CN109154027A (en) | 2016-04-15 | 2019-01-04 | 豪夫迈·罗氏有限公司 | For monitoring and the method for the treatment of cancer |
WO2017181098A2 (en) | 2016-04-15 | 2017-10-19 | Visterra, Inc. | Antibody molecules to zika virus and uses thereof |
CA3019921A1 (en) | 2016-04-15 | 2017-10-19 | Genentech, Inc. | Methods for monitoring and treating cancer |
BR112018071683A2 (en) | 2016-04-22 | 2019-02-19 | Obi Pharma, Inc. | method for treating breast cancer, method for treating a tumor in a patient, method for treating an individual suffering from cancer by immunotherapy, method for inducing / improving an immune response in an individual, method for improving obi-822 induced vaccine by immune response in an individual in need thereof, method for identifying a patient suitable for cancer therapy, and method for determining a patient's cancer treatment prognosis or drug response |
WO2017189730A1 (en) | 2016-04-26 | 2017-11-02 | Icahn School Of Medicine At Mount Sinai | Treatment of hippo pathway mutant tumors and methods of identifying subjects as candidates for treatment |
UA123323C2 (en) | 2016-05-02 | 2021-03-17 | Ф. Хоффманн-Ля Рош Аг | The contorsbody - a single chain target binder |
WO2017194441A1 (en) | 2016-05-11 | 2017-11-16 | F. Hoffmann-La Roche Ag | Modified anti-tenascin antibodies and methods of use |
SG11201809642TA (en) | 2016-05-16 | 2018-11-29 | Baxalta Inc | Anti-factor ix padua antibodies |
PL3458101T3 (en) | 2016-05-20 | 2021-05-31 | F. Hoffmann-La Roche Ag | Protac antibody conjugates and methods of use |
CN109415441B (en) | 2016-05-24 | 2023-04-07 | 英斯梅德股份有限公司 | Antibodies and methods of making same |
CN109313200B (en) | 2016-05-27 | 2022-10-04 | 豪夫迈·罗氏有限公司 | Bioanalytical methods for characterizing site-specific antibody-drug conjugates |
SG11201810023QA (en) | 2016-05-27 | 2018-12-28 | Agenus Inc | Anti-tim-3 antibodies and methods of use thereof |
EP3252078A1 (en) | 2016-06-02 | 2017-12-06 | F. Hoffmann-La Roche AG | Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer |
RU2745748C2 (en) | 2016-06-02 | 2021-03-31 | Эббви Инк. | Agonist of the glucocorticoid receptor and its immunoconjugates |
JP7043425B2 (en) | 2016-06-06 | 2022-03-29 | ジェネンテック, インコーポレイテッド | Silvestrol Antibodies-Drug Conjugates and Usage |
AU2017276604B2 (en) | 2016-06-06 | 2020-02-27 | F. Hoffmann-La Roche Ag | Fusion proteins for ophthalmology with increased eye retention |
EP3475298A1 (en) | 2016-06-24 | 2019-05-01 | H. Hoffnabb-La Roche Ag | Anti-polyubiquitin multispecific antibodies |
MA45491A (en) | 2016-06-27 | 2019-05-01 | Juno Therapeutics Inc | CMH-E RESTRICTED EPITOPES, BINDING MOLECULES AND RELATED METHODS AND USES |
EP3475446A1 (en) | 2016-06-27 | 2019-05-01 | Juno Therapeutics, Inc. | Method of identifying peptide epitopes, molecules that bind such epitopes and related uses |
EP3478717B1 (en) | 2016-07-04 | 2022-01-05 | F. Hoffmann-La Roche AG | Novel antibody format |
EP3481835A4 (en) | 2016-07-05 | 2020-02-26 | Blade Therapeutics, Inc. | Calpain modulators and therapeutic uses thereof |
KR20190039937A (en) | 2016-07-08 | 2019-04-16 | 스태튼 바이오테크놀로지 비.브이. | Anti-ApoC3 antibodies and methods of use thereof |
KR20190039134A (en) | 2016-07-13 | 2019-04-10 | 바이오젠 엠에이 인코포레이티드 | Use of LINGO-1 antagonists for the treatment regimens and for the treatment of dehydration diseases |
CN109715670A (en) | 2016-07-15 | 2019-05-03 | 波赛达治疗公司 | To the Chimeric antigen receptor (CAR) and its application method of MUC1 specificity |
WO2018014038A1 (en) | 2016-07-15 | 2018-01-18 | Poseida Therapeutics, Inc. | Chimeric antigen receptors and methods for use |
AU2017295886C1 (en) | 2016-07-15 | 2024-05-16 | Novartis Ag | Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor |
PL3496739T3 (en) | 2016-07-15 | 2021-10-11 | Acceleron Pharma Inc. | Compositions comprising actriia polypeptides for use in treating pulmonary hypertension |
WO2018014260A1 (en) | 2016-07-20 | 2018-01-25 | Nanjing Legend Biotech Co., Ltd. | Multispecific antigen binding proteins and methods of use thereof |
US20190330318A1 (en) | 2016-07-25 | 2019-10-31 | Biogen Ma Inc. | Anti-hspa5 (grp78) antibodies and uses thereof |
MX2019001043A (en) | 2016-07-27 | 2019-09-26 | Acceleron Pharma Inc | Methods and compositions for treating myelofibrosis. |
WO2018022933A1 (en) | 2016-07-27 | 2018-02-01 | Obi Pharma, Inc. | Immunogenic/therapeutic glycan compositions and uses thereof |
CN109415444B (en) | 2016-07-29 | 2024-03-01 | 中外制药株式会社 | Bispecific antibodies exhibiting increased functional activity of alternative FVIII cofactors |
US11643456B2 (en) | 2016-07-29 | 2023-05-09 | Obi Pharma, Inc. | Human antibodies, pharmaceutical compositions and methods |
WO2018026748A1 (en) | 2016-08-01 | 2018-02-08 | Xoma (Us) Llc | Parathyroid hormone receptor 1 (pth1r) antibodies and uses thereof |
US11858980B2 (en) | 2016-08-02 | 2024-01-02 | Visterra, Inc. | Engineered polypeptides and uses thereof |
WO2018027042A1 (en) | 2016-08-03 | 2018-02-08 | Bio-Techne Corporation | Identification of vsig3/vista as a novel immune checkpoint and use thereof for immunotherapy |
WO2018027124A1 (en) | 2016-08-05 | 2018-02-08 | Medimmune, Llc | Anti-o2 antibodies and uses thereof |
EP3494139B1 (en) | 2016-08-05 | 2022-01-12 | F. Hoffmann-La Roche AG | Multivalent and multiepitopic anitibodies having agonistic activity and methods of use |
KR20230079499A (en) | 2016-08-05 | 2023-06-07 | 추가이 세이야쿠 가부시키가이샤 | Composition for prophylaxis or treatment of il-8 related diseases |
CN109476748B (en) | 2016-08-08 | 2023-05-23 | 豪夫迈·罗氏有限公司 | Methods for treatment and diagnosis of cancer |
JP7093767B2 (en) | 2016-08-11 | 2022-06-30 | ジェネンテック, インコーポレイテッド | Pyrrolobenzodiazepine prodrug and its antibody conjugate |
CA3034057A1 (en) | 2016-08-22 | 2018-03-01 | CHO Pharma Inc. | Antibodies, binding fragments, and methods of use |
SG10201607778XA (en) | 2016-09-16 | 2018-04-27 | Chugai Pharmaceutical Co Ltd | Anti-Dengue Virus Antibodies, Polypeptides Containing Variant Fc Regions, And Methods Of Use |
US10751414B2 (en) | 2016-09-19 | 2020-08-25 | Celgene Corporation | Methods of treating psoriasis using PD-1 binding antibodies |
CN116731197A (en) | 2016-09-19 | 2023-09-12 | 豪夫迈·罗氏有限公司 | Complement factor based affinity chromatography |
EP3515943A4 (en) | 2016-09-19 | 2020-05-06 | Celgene Corporation | Methods of treating vitiligo using pd-1 binding proteins |
EP3515488A1 (en) | 2016-09-23 | 2019-07-31 | Teva Pharmaceuticals International GmbH | Treating cluster headache |
CN109952314A (en) | 2016-09-23 | 2019-06-28 | 泰瓦制药国际有限公司 | Treat intractable migraine |
CN118005799A (en) | 2016-09-23 | 2024-05-10 | 马伦戈治疗公司 | Multispecific antibody molecules comprising lambda light chain and kappa light chain |
FI3528838T3 (en) | 2016-09-23 | 2023-10-02 | Hoffmann La Roche | Uses of il-13 antagonists for treating atopic dermatitis |
CN110087681A (en) | 2016-09-28 | 2019-08-02 | 佐马美国有限公司 | The antibody of bind interleukin -2 and its purposes |
EP3523294A4 (en) | 2016-09-28 | 2021-01-13 | Blade Therapeutics, Inc. | Calpain modulators and therapeutic uses thereof |
GB201616596D0 (en) | 2016-09-29 | 2016-11-16 | Nascient Limited | Epitope and antibodies |
KR20190059305A (en) | 2016-09-30 | 2019-05-30 | 얀센 바이오테크 인코포레이티드 | A safe and effective way to treat psoriasis with anti-IL23-specific antibodies |
BR112019006706A2 (en) | 2016-10-03 | 2019-06-25 | Abbott Lab | improved methods for assessing gfap status in patient samples |
EP3522934A4 (en) | 2016-10-05 | 2020-04-15 | Acceleron Pharma Inc. | Compositions and method for treating kidney disease |
CN110139674B (en) | 2016-10-05 | 2023-05-16 | 豪夫迈·罗氏有限公司 | Method for preparing antibody drug conjugates |
KR20190072528A (en) | 2016-10-06 | 2019-06-25 | 제넨테크, 인크. | Treatment and Diagnosis Methods for Cancer |
TW202340473A (en) | 2016-10-07 | 2023-10-16 | 瑞士商諾華公司 | Treatment of cancer using chimeric antigen receptors |
WO2018068201A1 (en) | 2016-10-11 | 2018-04-19 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies and variants thereof against ctla-4 |
KR20230133934A (en) | 2016-10-11 | 2023-09-19 | 아게누스 인코포레이티드 | Anti-lag-3 antibodies and methods of use thereof |
WO2018071576A1 (en) | 2016-10-14 | 2018-04-19 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Treatment of tumors by inhibition of cd300f |
WO2018075408A1 (en) | 2016-10-17 | 2018-04-26 | Alexion Pharmaceuticals, Inc. | Methods of treating acute myeloid leukemia (aml) with combinations of anti-cd200 antibodies, cytarabine, and daunorubicin |
AU2017346488A1 (en) | 2016-10-19 | 2019-05-30 | Humabs Biomed Sa | Anti-O1 antibodies and uses thereof |
US11249082B2 (en) | 2016-10-29 | 2022-02-15 | University Of Miami | Zika virus assay systems |
EP3532091A2 (en) | 2016-10-29 | 2019-09-04 | H. Hoffnabb-La Roche Ag | Anti-mic antibidies and methods of use |
CA3039992A1 (en) | 2016-11-02 | 2018-05-11 | Jounce Therapeutics, Inc. | Antibodies to pd-1 and uses thereof |
EP3534957A1 (en) | 2016-11-02 | 2019-09-11 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and parp inhibitors |
WO2018083248A1 (en) | 2016-11-03 | 2018-05-11 | Kymab Limited | Antibodies, combinations comprising antibodies, biomarkers, uses & methods |
JP7045724B2 (en) | 2016-11-07 | 2022-04-01 | ニューラクル サイエンス カンパニー リミテッド | Anti-Family 19, member A5 antibody with sequence similarity and its uses |
AU2017362222A1 (en) | 2016-11-16 | 2019-05-30 | Janssen Biotech, Inc. | Method of treating psoriasis with anti-IL-23 specific antibody |
US11401330B2 (en) | 2016-11-17 | 2022-08-02 | Seagen Inc. | Glycan-interacting compounds and methods of use |
TW201829463A (en) | 2016-11-18 | 2018-08-16 | 瑞士商赫孚孟拉羅股份公司 | Anti-hla-g antibodies and use thereof |
WO2018094414A1 (en) | 2016-11-21 | 2018-05-24 | Obi Pharma, Inc. | Conjugated biological molecules, pharmaceutical compositions and methods |
WO2018098363A2 (en) | 2016-11-23 | 2018-05-31 | Bioverativ Therapeutics Inc. | Bispecific antibodies binding to coagulation factor ix and coagulation factor x |
CA3082255A1 (en) | 2016-11-23 | 2020-06-10 | Translational Drug Development, Llc | Benzamide and active compound compositions and methods of use |
WO2018102594A1 (en) | 2016-12-01 | 2018-06-07 | Alexion Pharmaceuticals, Inc. | Methods of treating solid tumors with anti-cd200 antibodies |
CN110300599A (en) | 2016-12-07 | 2019-10-01 | 艾吉纳斯公司 | Antibody and its application method |
KR102587130B1 (en) | 2016-12-07 | 2023-10-11 | 제넨테크, 인크. | Anti-tau antibodies and methods of using the same |
CN117820467A (en) | 2016-12-07 | 2024-04-05 | 基因泰克公司 | anti-TAU antibodies and methods of use |
AU2017373944B2 (en) | 2016-12-07 | 2022-02-03 | Agenus Inc. | Anti-CTLA-4 antibodies and methods of use thereof |
GB201621635D0 (en) | 2016-12-19 | 2017-02-01 | Ucb Biopharma Sprl | Crystal structure |
MX2019007411A (en) | 2016-12-21 | 2019-08-29 | Hoffmann La Roche | Re-use of enzymes in in vitro glycoengineering of antibodies. |
CN117887794A (en) | 2016-12-21 | 2024-04-16 | 豪夫迈·罗氏有限公司 | Methods for in vitro glycoengineering antibodies |
MX2019006266A (en) | 2016-12-21 | 2019-08-21 | Hoffmann La Roche | In vitro glycoengineering of antibodies. |
JP7360324B2 (en) | 2016-12-23 | 2023-10-12 | ビステラ, インコーポレイテッド | Binding polypeptides and methods of making them |
WO2018129329A1 (en) | 2017-01-06 | 2018-07-12 | Scholar Rock, Inc. | ISOFORM-SPECIFIC, CONTEXT-PERMISSIVE TGFβ1 INHIBITORS AND USE THEREOF |
WO2018129395A1 (en) | 2017-01-06 | 2018-07-12 | Scholar Rock, Inc. | Methods for treating metabolic diseases by inhibiting myostatin activation |
DK3565592T3 (en) | 2017-01-06 | 2023-05-01 | Scholar Rock Inc | TREATMENT OF METABOLIC DISEASES BY INHIBITION OF MYSTATIN ACTIVATION |
TW201831517A (en) | 2017-01-12 | 2018-09-01 | 美商優瑞科生物技術公司 | Constructs targeting histone h3 peptide/mhc complexes and uses thereof |
CN110290811A (en) | 2017-01-13 | 2019-09-27 | 中央研究院 | To treat the glue system of the repeatable loading of brain diseases |
US11135164B2 (en) | 2017-01-13 | 2021-10-05 | Academia Sinica | Reloadable hydrogel system comprising an anti-PEG antibody for treating myocardial infarction |
US11890319B2 (en) | 2017-01-18 | 2024-02-06 | Visterra, Inc. | Antibody molecule-drug conjugates and uses thereof |
WO2018136553A1 (en) | 2017-01-18 | 2018-07-26 | Genentech, Inc. | Idiotypic antibodies against anti-pd-l1 antibodies and uses thereof |
CN110234351A (en) | 2017-01-30 | 2019-09-13 | 詹森生物科技公司 | For treating the anti-TNF antibodies, composition and method of activity psoriatic arthritis |
US10240205B2 (en) | 2017-02-03 | 2019-03-26 | Population Bio, Inc. | Methods for assessing risk of developing a viral disease using a genetic test |
MX2019009359A (en) | 2017-02-06 | 2020-01-30 | Australian Meat & Live Stock | Immunostimulating compositions and uses therefore. |
JP2020506947A (en) | 2017-02-07 | 2020-03-05 | ヤンセン バイオテツク,インコーポレーテツド | Anti-TNF antibodies, compositions and methods for treating active ankylosing spondylitis |
US10266578B2 (en) | 2017-02-08 | 2019-04-23 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
WO2018148660A1 (en) | 2017-02-10 | 2018-08-16 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Neutralizing antibodies to plasmodium falciparum circumsporozoite protein and their use |
CR20190387A (en) | 2017-02-10 | 2019-09-25 | Genentech Inc | Anti-tryptase antibodies, compositions thereof, and uses thereof |
WO2018151820A1 (en) | 2017-02-16 | 2018-08-23 | Elstar Therapeutics, Inc. | Multifunctional molecules comprising a trimeric ligand and uses thereof |
WO2018152496A1 (en) | 2017-02-17 | 2018-08-23 | The Usa, As Represented By The Secretary, Dept. Of Health And Human Services | Compositions and methods for the diagnosis and treatment of zika virus infection |
SG11201907606XA (en) | 2017-02-27 | 2019-09-27 | Regeneron Pharma | Humanized model of kidney and liver disorders |
TW201837467A (en) | 2017-03-01 | 2018-10-16 | 美商建南德克公司 | Diagnostic and therapeutic methods for cancer |
AU2018226811A1 (en) | 2017-03-02 | 2019-09-26 | Beth Israel Deaconess Medical Center, Inc. | Selecting headache patients responsive to antibodies directed against calcitonin gene related peptide |
CN118063619A (en) | 2017-03-02 | 2024-05-24 | 诺华股份有限公司 | Engineered heterodimeric proteins |
JP2020510671A (en) | 2017-03-03 | 2020-04-09 | シアトル ジェネティックス, インコーポレイテッド | Glycan interacting compounds and methods of use |
KR20190124304A (en) | 2017-03-13 | 2019-11-04 | 포세이다 테라퓨틱스, 인크. | Compositions and Methods for Selective Removal and Replacement of Hematopoietic Stem Cells |
JP7227151B2 (en) | 2017-03-22 | 2023-02-21 | ジェネンテック, インコーポレイテッド | Antibody Compositions Optimized for Treatment of Eye Disorders |
AU2018240375C1 (en) | 2017-03-22 | 2024-02-01 | Ascendis Pharma A/S | Hydrogel cross-linked hyaluronic acid prodrug compositions and methods |
CA3052513A1 (en) | 2017-03-23 | 2018-09-27 | Abbott Laboratories | Methods for aiding in the diagnosis and determination of the extent of traumatic brain injury in a human subject using the early biomarker ubiquitin carboxy-terminal hydrolase l1 |
US20200132673A1 (en) | 2017-03-24 | 2020-04-30 | The Regents Of The University Of California | Proteoglycan irregularities in abnormal fibroblasts and therapies based therefrom |
KR20190133017A (en) | 2017-03-27 | 2019-11-29 | 에프. 호프만-라 로슈 아게 | Improved antigen binding receptor construction |
CR20190440A (en) | 2017-03-27 | 2019-11-12 | Hoffmann La Roche | Improved antigen binding receptors |
US11571459B2 (en) | 2017-04-03 | 2023-02-07 | Oncxerna Therapeutics, Inc. | Methods for treating cancer using PS-targeting antibodies with immuno-oncology agents |
CN110382525B (en) | 2017-04-03 | 2023-10-20 | 豪夫迈·罗氏有限公司 | Immunoconjugates |
AU2018247765B2 (en) | 2017-04-03 | 2023-11-23 | F. Hoffmann-La Roche Ag | Immunoconjugates of an Anti-PD-1 antibody with a mutant IL-2 or with IL-15 |
JP7247101B2 (en) | 2017-04-03 | 2023-03-28 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Antibody that binds to STEAP-1 |
IL268527B2 (en) | 2017-04-05 | 2024-03-01 | Hoffmann La Roche | Bispecific antibodies specifically binding to pd1 and lag3 |
WO2018185131A2 (en) | 2017-04-05 | 2018-10-11 | Novo Nordisk A/S | Oligomer extended insulin-fc conjugates |
LT3606954T (en) | 2017-04-05 | 2022-09-26 | F. Hoffmann-La Roche Ag | Anti-lag3 antibodies |
JP2020512825A (en) | 2017-04-12 | 2020-04-30 | ファイザー・インク | Antibodies with conditional affinity and methods of use thereof |
MX2019012223A (en) | 2017-04-13 | 2019-12-09 | Agenus Inc | Anti-cd137 antibodies and methods of use thereof. |
WO2018189379A1 (en) | 2017-04-14 | 2018-10-18 | Gamamabs Pharma | Amhrii-binding compounds for preventing or treating cancers |
MX2019012136A (en) | 2017-04-14 | 2020-07-20 | Gamamabs Pharma | Amhrii-binding compounds for preventing or treating lung cancers. |
CN110546513A (en) | 2017-04-15 | 2019-12-06 | 雅培实验室 | Methods for using early biomarkers to aid in hyperacute diagnosis and determination of traumatic brain injury in human subjects |
WO2018195283A1 (en) | 2017-04-19 | 2018-10-25 | Elstar Therapeutics, Inc. | Multispecific molecules and uses thereof |
MA49131A (en) | 2017-04-21 | 2020-03-25 | Hoffmann La Roche | USE OF KLK5 ANTAGONISTS FOR THE TREATMENT OF DISEASE |
AU2018255938A1 (en) | 2017-04-21 | 2019-10-31 | Staten Biotechnology B.V. | Anti-ApoC3 antibodies and methods of use thereof |
GB201706451D0 (en) | 2017-04-24 | 2017-06-07 | Imp Innovations Ltd | Cancer treatment |
EP3615569A1 (en) | 2017-04-25 | 2020-03-04 | The U.S.A. As Represented By The Secretary, Department Of Health And Human Services | Antibodies and methods for the diagnosis and treatment of epstein barr virus infection |
KR102663596B1 (en) | 2017-04-26 | 2024-05-10 | 유레카 쎄라퓨틱스, 인코포레이티드 | Construct that specifically recognizes glypican 3 and its use |
IL310182A (en) | 2017-04-26 | 2024-03-01 | Eureka Therapeutics Inc ׂ A Delaware Corp | Chimeric antibody/t-cell receptor constructs and uses thereof |
MX2019012793A (en) | 2017-04-27 | 2020-02-13 | Tesaro Inc | Antibody agents directed against lymphocyte activation gene-3 (lag-3) and uses thereof. |
EP3635407A1 (en) | 2017-04-28 | 2020-04-15 | Abbott Laboratories | Methods for aiding in the hyperacute diagnosis and determination of traumatic brain injury using early biomarkers on at least two samples from the same human subject |
CA3059769A1 (en) | 2017-04-28 | 2018-11-01 | Elstar Therapeutics, Inc. | Multispecific molecules comprising a non-immunoglobulin heterodimerization domain and uses thereof |
EP3618863B1 (en) | 2017-05-01 | 2023-07-26 | Agenus Inc. | Anti-tigit antibodies and methods of use thereof |
US10865238B1 (en) | 2017-05-05 | 2020-12-15 | Duke University | Complement factor H antibodies |
JOP20190256A1 (en) | 2017-05-12 | 2019-10-28 | Icahn School Med Mount Sinai | Newcastle disease viruses and uses thereof |
WO2018213097A1 (en) | 2017-05-15 | 2018-11-22 | University Of Rochester | Broadly neutralizing anti-influenza monoclonal antibody and uses thereof |
EP3630830A1 (en) | 2017-05-23 | 2020-04-08 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Novel cd73 antibody, preparation and uses thereof |
US10866251B2 (en) | 2017-05-25 | 2020-12-15 | Abbott Laboratories | Methods for aiding in the determination of whether to perform imaging on a human subject who has sustained or may have sustained an injury to the head using early biomarkers |
BR112019025387A2 (en) | 2017-05-30 | 2020-07-07 | Abbott Laboratories | methods to assist in the diagnosis and evaluation of a mild traumatic brain injury in a human subject using cardiac troponin i and early biomarkers |
US20210246227A1 (en) | 2017-05-31 | 2021-08-12 | Elstar Therapeutics, Inc. | Multispecific molecules that bind to myeloproliferative leukemia (mpl) protein and uses thereof |
AU2018277838A1 (en) | 2017-05-31 | 2019-12-19 | Stcube & Co., Inc. | Antibodies and molecules that immunospecifically bind to BTN1A1 and the therapeutic uses thereof |
KR20200024158A (en) | 2017-05-31 | 2020-03-06 | 주식회사 에스티큐브앤컴퍼니 | How to treat cancer using antibodies and molecules that immunospecifically bind to BTN1A1 |
CN111247167A (en) | 2017-06-02 | 2020-06-05 | 辉瑞公司 | Antibodies specific for FLT3 and uses thereof |
EP3630829A1 (en) | 2017-06-02 | 2020-04-08 | H. Hoffnabb-La Roche Ag | Type ii anti-cd20 antibody and anti-cd20/cd3 bispecific antibody for treatment of cancer |
MX2019013923A (en) | 2017-06-02 | 2020-01-21 | Pfizer | Chimeric antigen receptors targeting flt3. |
JP2020522562A (en) | 2017-06-06 | 2020-07-30 | ストキューブ アンド シーオー., インコーポレイテッド | Methods of treating cancer with antibodies and molecules that bind to BTN1A1 or BTN1A1 ligand |
WO2018226336A1 (en) | 2017-06-09 | 2018-12-13 | Providence Health & Services - Oregon | Utilization of cd39 and cd103 for identification of human tumor reactive cells for treatment of cancer |
GB201709379D0 (en) | 2017-06-13 | 2017-07-26 | Univ Leuven Kath | Humanised ADAMTS13 binding antibodies |
WO2018236904A1 (en) | 2017-06-19 | 2018-12-27 | Surface Oncology, Inc. | Combination of anti-cd47 antibodies and cell death-inducing agents, and uses thereof |
US11654135B2 (en) | 2017-06-22 | 2023-05-23 | Moonshot Pharma Llc | Methods for treating colon cancer with compositions comprising amlexanox and immune checkpoint inhibitors |
EP3642240A1 (en) | 2017-06-22 | 2020-04-29 | Novartis AG | Antibody molecules to cd73 and uses thereof |
US10752689B2 (en) | 2017-06-26 | 2020-08-25 | Bio-Techne Corporation | Hybridoma clones, monoclonal antibodies to VSIG-4, and methods of making and using |
CN111050791A (en) | 2017-06-27 | 2020-04-21 | 诺华股份有限公司 | Dosing regimens for anti-TIM-3 antibodies and uses thereof |
WO2019010131A1 (en) | 2017-07-03 | 2019-01-10 | Abbott Laboratories | Improved methods for measuring ubiquitin carboxy-terminal hydrolase l1 levels in blood |
WO2019010164A1 (en) | 2017-07-06 | 2019-01-10 | President And Fellows Of Harvard College | Evolution of trna synthetases |
TWI820031B (en) | 2017-07-11 | 2023-11-01 | 美商坎伯斯治療有限責任公司 | Agonist antibodies that bind human cd137 and uses thereof |
PE20200616A1 (en) | 2017-07-14 | 2020-03-11 | Pfizer | ANTIBODIES AGAINST MADCAM |
EP3431496A1 (en) | 2017-07-19 | 2019-01-23 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Anti- isoasp7 amyloid beta antibodies and uses thereof |
EP3655430A1 (en) | 2017-07-19 | 2020-05-27 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection |
US20200172617A1 (en) | 2017-07-20 | 2020-06-04 | Novartis Ag | Dosage regimens of anti-lag-3 antibodies and uses thereof |
US20190048073A1 (en) | 2017-07-20 | 2019-02-14 | Pfizer Inc. | Anti-gd3 antibodies and antibody-drug conjugates |
TWI823859B (en) | 2017-07-21 | 2023-12-01 | 美商建南德克公司 | Therapeutic and diagnostic methods for cancer |
WO2019023661A1 (en) | 2017-07-28 | 2019-01-31 | Scholar Rock, Inc. | Ltbp complex-specific inhibitors of tgf-beta 1 and uses thereof |
CR20200099A (en) | 2017-08-03 | 2020-07-24 | Amgen Inc | Interleukin-21 muteins and methods of treatment |
EP3589658A1 (en) | 2017-08-03 | 2020-01-08 | Alector LLC | Anti-cd33 antibodies and methods of use thereof |
WO2019035938A1 (en) | 2017-08-16 | 2019-02-21 | Elstar Therapeutics, Inc. | Multispecific molecules that bind to bcma and uses thereof |
WO2019036605A2 (en) | 2017-08-17 | 2019-02-21 | Massachusetts Institute Of Technology | Multiple specificity binders of cxc chemokines and uses thereof |
BR112020003533A2 (en) | 2017-08-25 | 2020-11-17 | Five Prime Therapeutics, Inc. | b7-h4 antibodies and methods of using them |
JP2020532987A (en) | 2017-09-08 | 2020-11-19 | ポセイダ セラピューティクス,インコーポレイティド | Compositions and Methods for Chimeric Ligand Receptor (CLR) -mediated Conditional Gene Expression |
SG11202001499WA (en) | 2017-09-08 | 2020-03-30 | Amgen Inc | Inhibitors of kras g12c and methods of using the same |
US11624130B2 (en) | 2017-09-18 | 2023-04-11 | President And Fellows Of Harvard College | Continuous evolution for stabilized proteins |
TW201922780A (en) | 2017-09-25 | 2019-06-16 | 美商健生生物科技公司 | Safe and effective method of treating Lupus with anti-IL12/IL23 antibody |
US11761963B2 (en) | 2017-09-27 | 2023-09-19 | Alexion Pharmaceuticals, Inc. | Biomarker signature for predicting tumor response to anti-CD200 therapy |
MA50667A (en) | 2017-09-29 | 2020-08-05 | Chugai Pharmaceutical Co Ltd | MULTISPECIFIC ANTIGEN-BINDING MOLECULE HAVING SUBSTITUTION ACTIVITY FOR THE FUNCTION OF BLOOD COAGULATION FACTOR VIII COFACTOR (FVIII), AND PHARMACEUTICAL FORMULATION CONTAINING THE SAME MOLECULE AS ACTIVE PRINCIPLE |
BR112020005419A2 (en) | 2017-10-02 | 2020-09-29 | Visterra, Inc. | antibody molecules for cd138 and their uses |
ES2759622T3 (en) | 2017-10-02 | 2020-05-11 | Certest Biotec S L | Anti-Dps antibodies and test devices for the detection of bacteria of the genus Campylobacter |
CA3078460A1 (en) | 2017-10-04 | 2019-04-11 | Opko Pharmaceuticals, Llc | Articles and methods directed to personalized therapy of cancer |
US11707522B2 (en) | 2017-10-13 | 2023-07-25 | Boehringer Ingelheim International Gmbh | Human antibodies to Tn antigen |
SG11202003486UA (en) | 2017-10-19 | 2020-05-28 | Debiopharm Int Sa | Combination product for the treatment of cancer |
US20200331975A1 (en) | 2017-10-20 | 2020-10-22 | Institut Curie | Dap10/12 based cars adapted for rush |
CN111315772A (en) | 2017-10-31 | 2020-06-19 | 斯塔顿生物技术有限公司 | anti-APOC 3 antibodies and methods of use thereof |
WO2019089594A1 (en) | 2017-10-31 | 2019-05-09 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and cytarabine |
MX2020004516A (en) | 2017-10-31 | 2020-10-20 | Allogene Therapeutics Inc | Methods and compositions for dosing of allogeneic chimeric antigen receptor t cells. |
US11718679B2 (en) | 2017-10-31 | 2023-08-08 | Compass Therapeutics Llc | CD137 antibodies and PD-1 antagonists and uses thereof |
JP2021500930A (en) | 2017-11-01 | 2021-01-14 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | COMP Body-Multivalent Target Binding Substance |
EP3704146B1 (en) | 2017-11-01 | 2021-12-15 | F. Hoffmann-La Roche AG | Trifab-contorsbody |
EP3707510B1 (en) | 2017-11-06 | 2024-06-26 | F. Hoffmann-La Roche AG | Diagnostic and therapeutic methods for cancer |
CN111587123A (en) | 2017-11-09 | 2020-08-25 | 品通治疗有限公司 | Methods and compositions for generating and using humanized conformation-specific phosphorylated tau antibodies |
CN111655288A (en) | 2017-11-16 | 2020-09-11 | 诺华股份有限公司 | Combination therapy |
US11851497B2 (en) | 2017-11-20 | 2023-12-26 | Compass Therapeutics Llc | CD137 antibodies and tumor antigen-targeting antibodies and uses thereof |
US11401345B2 (en) | 2017-11-27 | 2022-08-02 | Purdue Pharma L.P. | Humanized antibodies targeting human tissue factor |
KR20200090823A (en) | 2017-11-30 | 2020-07-29 | 제넨테크, 인크. | Anti-PD-L1 antibody and method of using the same antibody for PD-L1 detection |
WO2019113464A1 (en) | 2017-12-08 | 2019-06-13 | Elstar Therapeutics, Inc. | Multispecific molecules and uses thereof |
JP7379165B2 (en) | 2017-12-09 | 2023-11-14 | アボット・ラボラトリーズ | Methods for aiding in diagnosing and assessing traumatic brain injury in human subjects using a combination of GFAP and UCH-L1 |
JP7344801B2 (en) | 2017-12-09 | 2023-09-14 | アボット・ラボラトリーズ | Glial fibrillary acidic protein (GFAP) and/or ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) Methods to assist in diagnosis and assessment of injuries sustained or potential injuries |
WO2019126536A1 (en) | 2017-12-20 | 2019-06-27 | Alexion Pharmaceuticals Inc. | Humanized anti-cd200 antibodies and uses thereof |
US20210087267A1 (en) | 2017-12-20 | 2021-03-25 | Alexion Pharmaceuticals, Inc. | Liquid formulations of anti-cd200 antibodies |
JP7394058B2 (en) | 2017-12-21 | 2023-12-07 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Universal reporter cell assay for specificity testing of novel antigen-binding moieties |
AU2018390881A1 (en) | 2017-12-21 | 2020-07-02 | F. Hoffmann-La Roche Ag | Antibodies binding to HLA-A2/WT1 |
CN111492243A (en) | 2017-12-21 | 2020-08-04 | 豪夫迈·罗氏有限公司 | CAR-T cell assay for specific testing of novel antigen binding modules |
KR20200103706A (en) | 2017-12-22 | 2020-09-02 | 조운스 테라퓨틱스, 인크. | Antibodies to LILRB2 |
US20190211098A1 (en) | 2017-12-22 | 2019-07-11 | Genentech, Inc. | Use of pilra binding agents for treatment of a disease |
TW201930350A (en) | 2017-12-28 | 2019-08-01 | 大陸商南京傳奇生物科技有限公司 | Antibodies and variants thereof against PD-L1 |
KR20200104333A (en) | 2017-12-28 | 2020-09-03 | 난징 레전드 바이오테크 씨오., 엘티디. | Single-domain antibodies to TIGIT and variants thereof |
EP3732193A1 (en) | 2017-12-29 | 2020-11-04 | Alector LLC | Anti-tmem106b antibodies and methods of use thereof |
UY38049A (en) | 2018-01-12 | 2019-07-31 | Amgen Inc | ANTI-PD-1 ANTIBODIES AND TREATMENT METHODS |
EP3737696A1 (en) | 2018-01-12 | 2020-11-18 | Bristol-Myers Squibb Company | Combination therapy with anti-il-8 antibodies and anti-pd-1 antibodies for treating cancer |
CA3084518A1 (en) | 2018-01-15 | 2019-07-18 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies and variants thereof against pd-1 |
US20200339686A1 (en) | 2018-01-16 | 2020-10-29 | Lakepharma, Inc. | Bispecific antibody that binds cd3 and another target |
WO2019152715A1 (en) | 2018-01-31 | 2019-08-08 | Alector Llc | Anti-ms4a4a antibodies and methods of use thereof |
TW201934580A (en) | 2018-02-01 | 2019-09-01 | 美商輝瑞大藥廠 | Antibodies specific for CD70 and their uses |
BR112020015662A2 (en) | 2018-02-01 | 2020-12-08 | Pfizer Inc. | CHEMICAL ANTIGEN RECEPTORS AIMED AT CD70 |
WO2019152810A1 (en) | 2018-02-02 | 2019-08-08 | Bio-Techne Corporation | Compounds that modulate the interaction of vista and vsig3 and methods of making and using |
WO2019150309A1 (en) | 2018-02-02 | 2019-08-08 | Hammack Scott | Modulators of gpr68 and uses thereof for treating and preventing diseases |
AU2019218786B2 (en) | 2018-02-07 | 2024-05-02 | Dana-Farber Cancer Institute, Inc. | Cell-permeable stapled peptide modules for cellular delivery |
EP3749361A1 (en) | 2018-02-08 | 2020-12-16 | F. Hoffmann-La Roche AG | Bispecific antigen-binding molecules and methods of use |
KR20230142806A (en) | 2018-02-09 | 2023-10-11 | 제넨테크, 인크. | Therapeutic and diagnostic methods for mast cell-mediated inflammatory diseases |
TWI829667B (en) | 2018-02-09 | 2024-01-21 | 瑞士商赫孚孟拉羅股份公司 | Antibodies binding to gprc5d |
EP3752195A4 (en) | 2018-02-14 | 2021-11-17 | Viela Bio, Inc. | Antibodies to feline mcdonough sarcoma (fms)-like tyrosine kinase 3 receptor ligand (flt3l) and uses thereof for treating autoimmune and inflammatory diseases |
GB201802486D0 (en) | 2018-02-15 | 2018-04-04 | Ucb Biopharma Sprl | Methods |
CA3092108A1 (en) | 2018-02-26 | 2019-08-29 | Genentech, Inc. | Dosing for treatment with anti-tigit and anti-pd-l1 antagonist antibodies |
EP3759491A1 (en) | 2018-03-01 | 2021-01-06 | H. Hoffnabb-La Roche Ag | Specificity assay for novel target antigen binding moieties |
CA3091801A1 (en) | 2018-03-02 | 2019-09-06 | Five Prime Therapeutics, Inc. | B7-h4 antibodies and methods of use thereof |
MX2020009265A (en) | 2018-03-05 | 2020-10-01 | Janssen Biotech Inc | Methods of treating crohn's disease with anti-il23 specific antibody. |
BR112020017701A2 (en) | 2018-03-12 | 2020-12-29 | Zoetis Services Llc | ANTI-NGF ANTIBODIES AND METHODS OF THE SAME |
WO2019178362A1 (en) | 2018-03-14 | 2019-09-19 | Elstar Therapeutics, Inc. | Multifunctional molecules that bind to calreticulin and uses thereof |
WO2019178364A2 (en) | 2018-03-14 | 2019-09-19 | Elstar Therapeutics, Inc. | Multifunctional molecules and uses thereof |
US20200040103A1 (en) | 2018-03-14 | 2020-02-06 | Genentech, Inc. | Anti-klk5 antibodies and methods of use |
JP7037218B2 (en) | 2018-03-14 | 2022-03-16 | サーフィス オンコロジー インコーポレイテッド | Antibodies that bind to CD39 and their use |
CN116327926A (en) | 2018-03-15 | 2023-06-27 | 中外制药株式会社 | Anti-dengue virus antibodies with cross-reactivity to Zika virus and methods of use |
CA3093740A1 (en) | 2018-03-22 | 2019-09-26 | Surface Oncology, Inc. | Anti-il-27 antibodies and uses thereof |
WO2019180272A1 (en) | 2018-03-23 | 2019-09-26 | Fundación Instituto De Investigación Sanitaria De Santiago De Compostela | Anti-leptin affinity reagents for use in the treatment of obesity and other leptin-resistance associated diseases |
EP3775900A1 (en) | 2018-03-26 | 2021-02-17 | Alexion Pharmaceuticals, Inc. | High throughput method for measuring the protease activity of complement c3 convertase |
EP3775909B1 (en) | 2018-03-26 | 2023-05-10 | Glycanostics s.r.o. | Means and methods for glycoprofiling of a protein |
US11958903B2 (en) | 2018-03-30 | 2024-04-16 | Nanjing Legend Biotech Co., Ltd. | Single-domain antibodies against LAG-3 and uses thereof |
PE20201343A1 (en) | 2018-04-02 | 2020-11-25 | Bristol Myers Squibb Co | ANTI-TREM-1 ANTIBODIES AND USES OF THEM |
JP7104458B2 (en) | 2018-04-02 | 2022-07-21 | 上海博威生物医薬有限公司 | Lymphocyte activation gene-3 (LAG-3) -binding antibody and its use |
WO2019192973A1 (en) | 2018-04-04 | 2019-10-10 | F. Hoffmann-La Roche Ag | Diagnostic assays to detect tumor antigens in cancer patients |
TW202011029A (en) | 2018-04-04 | 2020-03-16 | 美商建南德克公司 | Methods for detecting and quantifying FGF21 |
JP2021520209A (en) | 2018-04-04 | 2021-08-19 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Diagnostic Assay for Detecting Tumor Antigens in Cancer Patients |
WO2019200357A1 (en) | 2018-04-12 | 2019-10-17 | Surface Oncology, Inc. | Biomarker for cd47 targeting therapeutics and uses therefor |
US20210147547A1 (en) | 2018-04-13 | 2021-05-20 | Novartis Ag | Dosage Regimens For Anti-Pd-L1 Antibodies And Uses Thereof |
AR114789A1 (en) | 2018-04-18 | 2020-10-14 | Hoffmann La Roche | ANTI-HLA-G ANTIBODIES AND THE USE OF THEM |
AR115052A1 (en) | 2018-04-18 | 2020-11-25 | Hoffmann La Roche | MULTI-SPECIFIC ANTIBODIES AND THE USE OF THEM |
WO2019207159A1 (en) | 2018-04-27 | 2019-10-31 | Fondazione Ebri Rita Levi-Montalcini | Antibody directed against a tau-derived neurotoxic peptide and uses thereof |
EP3787678A1 (en) | 2018-05-03 | 2021-03-10 | University Of Rochester | Anti-influenza neuraminidase monoclonal antibodies and uses thereof |
AU2019265888A1 (en) | 2018-05-10 | 2020-11-26 | Neuracle Science Co., Ltd. | Anti-family with sequence similarity 19, member A5 antibodies and method of use thereof |
MX2020012081A (en) | 2018-05-11 | 2021-04-28 | Wuxi Biologics Shanghai Co Ltd | Fully human antibodies against ox40, method for preparing same, and use thereof. |
EP3790587A4 (en) | 2018-05-11 | 2022-01-26 | Janssen Biotech, Inc. | Methods of treating depression using il-23 antibodies |
WO2019222130A1 (en) | 2018-05-15 | 2019-11-21 | Immunogen, Inc. | Combination treatment with antibody-drug conjugates and flt3 inhibitors |
CA3099308A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Compositions and methods for enhancing the killing of target cells by nk cells |
WO2019226658A1 (en) | 2018-05-21 | 2019-11-28 | Compass Therapeutics Llc | Multispecific antigen-binding compositions and methods of use |
TWI803637B (en) | 2018-05-23 | 2023-06-01 | 美商輝瑞大藥廠 | Antibodies specific for gucy2c and uses thereof |
AU2019274654B2 (en) | 2018-05-23 | 2023-07-20 | Pfizer Inc. | Antibodies specific for CD3 and uses thereof |
CR20200566A (en) | 2018-05-25 | 2021-02-19 | Alector Llc | Anti-sirpa antibodies and methods of use thereof |
UY38247A (en) | 2018-05-30 | 2019-12-31 | Novartis Ag | ANTIBODIES AGAINST ENTPD2, COMBINATION THERAPIES AND METHODS OF USE OF ANTIBODIES AND COMBINATION THERAPIES |
BR112020023505A2 (en) | 2018-06-01 | 2021-03-30 | Tayu Huaxia Biotech Medical Group Co., Ltd. | COMPOSITIONS AND USES OF THE SAME FOR TREATING DISEASE OR CONDITION |
US11830582B2 (en) | 2018-06-14 | 2023-11-28 | University Of Miami | Methods of designing novel antibody mimetics for use in detecting antigens and as therapeutic agents |
US11913044B2 (en) | 2018-06-14 | 2024-02-27 | President And Fellows Of Harvard College | Evolution of cytidine deaminases |
CN112533632A (en) | 2018-06-18 | 2021-03-19 | Ucb生物制药有限责任公司 | GREMLIN-1 antagonists for the prevention and treatment of cancer |
US11993661B2 (en) | 2018-06-18 | 2024-05-28 | Eureka Therapeutics, Inc. | Constructs targeting prostate-specific membrane antigen (PSMA) and uses thereof |
WO2019245817A1 (en) | 2018-06-19 | 2019-12-26 | Armo Biosciences, Inc. | Compositions and methods of use of il-10 agents in conjunction with chimeric antigen receptor cell therapy |
WO2019246293A2 (en) | 2018-06-19 | 2019-12-26 | Atarga, Llc | Antibody molecules to complement component 5 and uses thereof |
TW202016144A (en) | 2018-06-21 | 2020-05-01 | 日商第一三共股份有限公司 | Compositions including cd3 antigen binding fragments and uses thereof |
CA3103017A1 (en) | 2018-06-23 | 2019-12-26 | Genentech, Inc. | Methods of treating lung cancer with a pd-1 axis binding antagonist, a platinum agent, and a topoisomerase ii inhibitor |
WO2020003172A1 (en) | 2018-06-26 | 2020-01-02 | Mor Research Applications | Transthyretin antibodies and uses thereof |
TW202035444A (en) | 2018-06-27 | 2020-10-01 | 台灣浩鼎生技股份有限公司 | Glycosynthase variants for glycoprotein engineering and methods of use |
EP3814377A2 (en) | 2018-06-29 | 2021-05-05 | Alector LLC | Anti-sirp-beta1 antibodies and methods of use thereof |
EP3818083A2 (en) | 2018-07-03 | 2021-05-12 | Elstar Therapeutics, Inc. | Anti-tcr antibody molecules and uses thereof |
WO2020008083A1 (en) | 2018-07-05 | 2020-01-09 | Consejo Superior De Investigaciones Científicas | Therapeutic target in chemokine receptors for the screening of compounds useful for the treatment of pathological processes involving chemokine signaling |
JP2021531254A (en) | 2018-07-11 | 2021-11-18 | スカラー ロック インコーポレイテッドScholar Rock, Inc. | High affinity isoform-selective TGFβ1 inhibitor, and its use |
ES2905160T3 (en) | 2018-07-11 | 2022-04-07 | Scholar Rock Inc | Selective inhibitors of the TGFBeta1 isoform and their use |
US20210340238A1 (en) | 2018-07-11 | 2021-11-04 | Scholar Rock, Inc. | TGFß1 INHIBITORS AND USE THEREOF |
CN112334479A (en) | 2018-07-13 | 2021-02-05 | 南京传奇生物科技有限公司 | Co-receptor systems for the treatment of infectious diseases |
KR20230065382A (en) | 2018-07-13 | 2023-05-11 | 알렉터 엘엘씨 | Anti-sortilin antibodies and methods of use thereof |
MX2021000558A (en) | 2018-07-18 | 2021-04-13 | Genentech Inc | Methods of treating lung cancer with a pd-1 axis binding antagonist, an antimetabolite, and a platinum agent. |
JP2021530697A (en) | 2018-07-18 | 2021-11-11 | ヤンセン バイオテツク,インコーポレーテツド | Sustained response predictor after treatment with anti-IL23 specific antibody |
CN112740043A (en) | 2018-07-20 | 2021-04-30 | 皮埃尔法布雷医药公司 | VISTA receptor |
WO2020021465A1 (en) | 2018-07-25 | 2020-01-30 | Advanced Accelerator Applications (Italy) S.R.L. | Method of treatment of neuroendocrine tumors |
EP3625368B8 (en) | 2018-08-08 | 2022-12-14 | PML Screening, LLC | Methods for assessing the risk of developing progressive multifocal leukoencephalopathy caused by john cunningham virus by genetic testing |
MX2021001453A (en) | 2018-08-08 | 2021-04-12 | Genentech Inc | Use of tryptophan derivatives and l-methionine for protein formulation. |
WO2020033926A2 (en) | 2018-08-09 | 2020-02-13 | Compass Therapeutics Llc | Antibodies that bind cd277 and uses thereof |
EP3833443A1 (en) | 2018-08-09 | 2021-06-16 | Compass Therapeutics LLC | Antigen binding agents that bind cd277 and uses thereof |
WO2020033925A2 (en) | 2018-08-09 | 2020-02-13 | Compass Therapeutics Llc | Antibodies that bind cd277 and uses thereof |
AU2019317229A1 (en) | 2018-08-10 | 2021-02-18 | Eutilex Co.,Ltd | Chimeric Antigen Receptor that Binds HLA-DR, and CAR-T Cell |
BR112021002037A2 (en) | 2018-08-10 | 2021-05-04 | Chugai Seiyaku Kabushiki Kaisha | anti-cd137 antigen binding molecule and its use |
WO2020041360A1 (en) | 2018-08-21 | 2020-02-27 | Quidel Corporation | Dbpa antibodies and uses thereof |
AU2019331018A1 (en) | 2018-08-31 | 2021-03-11 | Alector Llc | Anti-cd33 antibodies and methods of use thereof |
GB201814281D0 (en) | 2018-09-03 | 2018-10-17 | Femtogenix Ltd | Cytotoxic agents |
US10899826B1 (en) | 2018-09-13 | 2021-01-26 | Teva Pharmaceuticals International Gmbh | Pharmaceutical compositions for an anti-CGRP antagonist antibody |
WO2020061060A1 (en) | 2018-09-19 | 2020-03-26 | Genentech, Inc. | Therapeutic and diagnostic methods for bladder cancer |
CA3111809A1 (en) | 2018-09-21 | 2020-03-26 | Genentech, Inc. | Diagnostic methods for triple-negative breast cancer |
CA3160103A1 (en) | 2018-09-24 | 2020-04-02 | Janssen Biotech, Inc. | Safe and effective method of treating ulcerative colitis with anti-il12/il23 antibody |
AU2019346645A1 (en) | 2018-09-27 | 2021-04-29 | Marengo Therapeutics, Inc. | CSF1R/CCR2 multispecific antibodies |
CA3114295A1 (en) | 2018-09-28 | 2020-04-02 | Kyowa Kirin Co., Ltd. | Il-36 antibodies and uses thereof |
CN112969503A (en) | 2018-10-03 | 2021-06-15 | 斯塔滕生物技术有限公司 | Antibodies specific for human and cynomolgus macaques APOC3 and methods of use thereof |
WO2020070303A1 (en) | 2018-10-05 | 2020-04-09 | Bavarian Nordic A/S | Combination therapy for treating cancer with an intravenous administration of a recombinant mva and an immune checkpoint antagonist or agonist |
AU2019359540A1 (en) | 2018-10-11 | 2021-04-29 | Pfizer Inc. | Dosage regimen for TFPI antagonists |
UY38407A (en) | 2018-10-15 | 2020-05-29 | Novartis Ag | TREM2 STABILIZING ANTIBODIES |
CA3116324A1 (en) | 2018-10-18 | 2020-04-23 | Genentech, Inc. | Diagnostic and therapeutic methods for sarcomatoid kidney cancer |
SG11202104010PA (en) | 2018-10-23 | 2021-05-28 | Scholar Rock Inc | Rgmc-selective inhibitors and use thereof |
GB201817311D0 (en) | 2018-10-24 | 2018-12-05 | Ucb Biopharma Sprl | Antibodies |
WO2020086858A1 (en) | 2018-10-24 | 2020-04-30 | Genentech, Inc. | Conjugated chemical inducers of degradation and methods of use |
GB201817309D0 (en) | 2018-10-24 | 2018-12-05 | Ucb Biopharma Sprl | Antibodies |
WO2020086408A1 (en) | 2018-10-26 | 2020-04-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | A high-yield perfusion-based transient gene expression bioprocess |
JP2022506156A (en) | 2018-11-05 | 2022-01-17 | ジェネンテック, インコーポレイテッド | Method for producing double-stranded protein in prokaryotic host cell |
KR20210101235A (en) | 2018-11-16 | 2021-08-18 | 메모리얼 슬로안 케터링 캔서 센터 | Antibodies to mucin-16 and methods of use thereof |
SG11202104918PA (en) | 2018-11-20 | 2021-06-29 | Bavarian Nordic As | Therapy for treating cancer with an intratumoral and/or intravenous administration of a recombinant mva encoding 4-1bbl (cd137l) and/or cd40l |
AU2019383017A1 (en) | 2018-11-20 | 2021-06-03 | Janssen Biotech, Inc. | Safe and effective method of treating psoriasis with anti-IL-23 specific antibody |
US20220332800A1 (en) | 2018-11-20 | 2022-10-20 | Takeda Vaccines, Inc. | Novel anti-zika virus antibodies and uses thereof |
WO2020118011A1 (en) | 2018-12-06 | 2020-06-11 | Alexion Pharmaceuticals, Inc. | Anti-alk2 antibodies and uses thereof |
AU2018451747A1 (en) | 2018-12-06 | 2021-06-17 | F. Hoffmann-La Roche Ag | Combination therapy of diffuse large B-cell lymphoma comprising an anti-CD79b immunoconjugates, an alkylating agent and an anti-CD20 antibody |
WO2020123275A1 (en) | 2018-12-10 | 2020-06-18 | Genentech, Inc. | Photocrosslinking peptides for site specific conjugation to fc-containing proteins |
EP3897722A4 (en) | 2018-12-18 | 2022-09-14 | Janssen Biotech, Inc. | Safe and effective method of treating lupus with anti-il12/il23 antibody |
JP2022514290A (en) | 2018-12-20 | 2022-02-10 | ジェネンテック, インコーポレイテッド | Modified antibody FC and usage |
WO2020132214A2 (en) | 2018-12-20 | 2020-06-25 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Ebola virus glycoprotein-specific monoclonal antibodies and uses thereof |
US20220042038A1 (en) | 2018-12-20 | 2022-02-10 | Poseida Therapeutics, Inc. | Nanotransposon compositions and methods of use |
EP3897648B1 (en) | 2018-12-20 | 2023-08-23 | Novartis AG | Extended low dose regimens for mdm2 inhibitors |
AU2019402163A1 (en) | 2018-12-20 | 2021-07-08 | Kyowa Kirin Co., Ltd. | Fn14 antibodies and uses thereof |
WO2020132231A1 (en) | 2018-12-21 | 2020-06-25 | Genentech, Inc. | Methods of producing polypeptides using a cell line resistant to apoptosis |
TW202039552A (en) | 2018-12-21 | 2020-11-01 | 瑞士商赫孚孟拉羅股份公司 | Antibody that binds to vegf and il-1beta and methods of use |
JP2022516505A (en) | 2018-12-28 | 2022-02-28 | スパークス・セラピューティクス・インコーポレイテッド | Claudin 18.2 specific binding molecule, composition and method thereof for the treatment of cancer and other diseases. |
EP3902560A1 (en) | 2018-12-28 | 2021-11-03 | F. Hoffmann-La Roche AG | A peptide-mhc-i-antibody fusion protein for therapeutic use in a patient with amplified immune response |
CA3126654A1 (en) | 2019-01-15 | 2020-07-23 | Janssen Biotech, Inc. | Anti-tnf antibody compositions and methods for the treatment of juvenile idiopathic arthritis |
MX2021008453A (en) | 2019-01-16 | 2021-08-19 | Compass Therapeutics Llc | Formulations of antibodies that bind human cd137 and uses thereof. |
GB201900732D0 (en) | 2019-01-18 | 2019-03-06 | Ucb Biopharma Sprl | Antibodies |
MX2021008871A (en) | 2019-01-23 | 2021-08-19 | Janssen Biotech Inc | Anti-tnf antibody compositions for use in methods for the treatment of psoriatic arthritis. |
JP2022523475A (en) | 2019-01-23 | 2022-04-25 | ジェネンテック, インコーポレイテッド | How to produce multimeric proteins in eukaryotic host cells |
CN113329770A (en) | 2019-01-24 | 2021-08-31 | 中外制药株式会社 | Novel cancer antigen and antibody against said antigen |
CN113348178A (en) | 2019-01-28 | 2021-09-03 | 枫叶生物技术有限公司 | PSMP antagonists for the treatment of fibrotic diseases of the lung, kidney or liver |
GB201901197D0 (en) | 2019-01-29 | 2019-03-20 | Femtogenix Ltd | G-A Crosslinking cytotoxic agents |
MX2021009175A (en) | 2019-01-30 | 2021-09-14 | Scholar Rock Inc | LTBP COMPLEX-SPECIFIC INHIBITORS OF TGFß AND USES THEREOF. |
US11738050B2 (en) | 2019-02-01 | 2023-08-29 | Regents Of The University Of Minnesota | Compounds binding to fibroblast activation protein alpha |
EP3693063A1 (en) | 2019-02-06 | 2020-08-12 | Diaccurate | Methods and compositions for treating cancer |
EP3696191A1 (en) | 2019-02-14 | 2020-08-19 | Fundación Instituto de Investigación contra la Leucemia Josep Carreras (IJC) | Car t-cells for the treatment of cd1a-positive cancer |
US10871640B2 (en) | 2019-02-15 | 2020-12-22 | Perkinelmer Cellular Technologies Germany Gmbh | Methods and systems for automated imaging of three-dimensional objects |
JP2022521937A (en) | 2019-02-21 | 2022-04-13 | マレンゴ・セラピューティクス,インコーポレーテッド | Antibody molecules that bind to NKp30 and their use |
CN114026122A (en) | 2019-02-21 | 2022-02-08 | 马伦戈治疗公司 | Multifunctional molecules that bind to T cell-associated cancer cells and uses thereof |
CN114127112A (en) | 2019-02-21 | 2022-03-01 | 马伦戈治疗公司 | Multifunctional molecules that bind to T cells and their use to treat autoimmune disorders |
SG11202109056TA (en) | 2019-02-21 | 2021-09-29 | Marengo Therapeutics Inc | Multifunctional molecules that bind to calreticulin and uses thereof |
AU2020226904A1 (en) | 2019-02-21 | 2021-09-16 | Marengo Therapeutics, Inc. | Anti-TCR antibody molecules and uses thereof |
MA55080A (en) | 2019-02-26 | 2022-01-05 | Inspirna Inc | HIGH AFFINITY ANTI-MERTK ANTIBODIES AND ASSOCIATED USES |
JP2022521773A (en) | 2019-02-27 | 2022-04-12 | ジェネンテック, インコーポレイテッド | Dosing for treatment with anti-TIGIT antibody and anti-CD20 antibody or anti-CD38 antibody |
KR20210138588A (en) | 2019-03-08 | 2021-11-19 | 제넨테크, 인크. | Methods for detecting and quantifying membrane-associated proteins on extracellular vesicles |
CA3133395A1 (en) | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
JP2022525179A (en) | 2019-03-14 | 2022-05-11 | ヤンセン バイオテツク,インコーポレーテツド | Production method for producing anti-TNF antibody composition |
EP3938391A1 (en) | 2019-03-14 | 2022-01-19 | Janssen Biotech, Inc. | Methods for producing anti-tnf antibody compositions |
US20220153829A1 (en) | 2019-03-14 | 2022-05-19 | Janssen Biotech, Inc. | Methods for Producing Anti-TNF Antibody Compositions |
CN113853385A (en) | 2019-03-18 | 2021-12-28 | 詹森生物科技公司 | Methods of treating psoriasis in pediatric subjects with anti-IL 12/IL23 antibodies |
EP3947442A2 (en) | 2019-03-28 | 2022-02-09 | Danisco US Inc. | Engineered antibodies |
SG11202110732XA (en) | 2019-03-29 | 2021-10-28 | Atarga Llc | Anti fgf23 antibody |
WO2020205626A1 (en) | 2019-03-29 | 2020-10-08 | Genentech, Inc. | Modulators of cell surface protein interactions and methods and compositions related to same |
KR20220002967A (en) | 2019-04-19 | 2022-01-07 | 제넨테크, 인크. | Anti-MERTK antibodies and methods of use thereof |
EP3957325A4 (en) | 2019-04-19 | 2023-01-04 | Chugai Seiyaku Kabushiki Kaisha | Chimeric receptor recognizing modification site of antibody |
US20220289854A1 (en) | 2019-04-30 | 2022-09-15 | Dana-Farber Cancer Institute, Inc. | Methods for treating cancer using combinations of anti-cx3cr1 and immune checkpoint blockade agents |
EP3962523A2 (en) | 2019-05-03 | 2022-03-09 | The United States of America, as represented by the Secretary, Department of Health and Human Services | Neutralizing antibodies to plasmodium falciparum circumsporozoite protein and their use |
KR20220007136A (en) | 2019-05-14 | 2022-01-18 | 제넨테크, 인크. | Methods of Use of Anti-CD79b Immunoconjugates to Treat Follicular Lymphoma |
US20230085439A1 (en) | 2019-05-21 | 2023-03-16 | University Of Georgia Research Foundation, Inc. | Antibodies that bind human metapneumovirus fusion protein and their use |
BR112021023295A2 (en) | 2019-05-23 | 2022-02-08 | Janssen Biotech Inc | Method of treating inflammatory bowel disease with a combination therapy of antibodies to il-23 and tnf-alpha |
AU2020281535A1 (en) | 2019-05-24 | 2022-01-27 | Merck Patent Gmbh | Combination therapies using CDK inhibitors |
WO2020245676A1 (en) | 2019-06-03 | 2020-12-10 | Janssen Biotech, Inc. | Anti-tnf antibody compositions, and methods for the treatment of psoriatic arthritis |
KR20220030952A (en) | 2019-06-03 | 2022-03-11 | 얀센 바이오테크 인코포레이티드 | Anti-TNF antibodies, compositions, and methods for the treatment of active ankylosing spondylitis |
JP2022536839A (en) | 2019-06-11 | 2022-08-19 | アレクトル エルエルシー | Anti-sortilin antibody for therapeutic use |
EP3983442A2 (en) | 2019-06-17 | 2022-04-20 | Visterra, Inc. | Humanized antibody molecules to cd138 and uses thereof |
WO2021001289A1 (en) | 2019-07-02 | 2021-01-07 | F. Hoffmann-La Roche Ag | Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody |
CN114341187A (en) | 2019-07-12 | 2022-04-12 | 中外制药株式会社 | Anti-mutant FGFR3 antibodies and uses thereof |
AR119393A1 (en) | 2019-07-15 | 2021-12-15 | Hoffmann La Roche | ANTIBODIES THAT BIND NKG2D |
US20220306734A1 (en) | 2019-07-24 | 2022-09-29 | H. Lundbeck A/S | Anti-mglur5 antibodies and uses thereof |
BR112022001415A2 (en) | 2019-07-26 | 2022-06-07 | Visterra Inc | Interleukin-2 agents and their uses |
CN114174338A (en) | 2019-07-31 | 2022-03-11 | 豪夫迈·罗氏有限公司 | Antibodies that bind to GPRC5D |
CR20220078A (en) | 2019-07-31 | 2022-06-24 | Alector Llc | Anti-ms4a4a antibodies and methods of use thereof |
EP4003526A2 (en) | 2019-07-31 | 2022-06-01 | F. Hoffmann-La Roche AG | Antibodies binding to gprc5d |
HUE064890T2 (en) | 2019-08-02 | 2024-04-28 | Fundacio De Recerca Clinic Barcelona Inst | Car t-cells against bcma for the treatment of multiple myeloma |
CA3148740A1 (en) | 2019-08-06 | 2021-02-11 | Aprinoia Therapeutics Limited | Antibodies that bind to pathological tau species and uses thereof |
WO2021023860A1 (en) | 2019-08-07 | 2021-02-11 | Db Biotech, As | Improved horseradish peroxidase polypeptides |
CN114867751A (en) | 2019-08-12 | 2022-08-05 | 阿帕特夫研究和发展有限公司 | 4-1BB and OX40 binding proteins and related compositions and methods, anti-4-1 BB antibodies, anti-OX 40 antibodies |
BR112022002406A2 (en) | 2019-08-12 | 2022-07-19 | Purinomia Biotech Inc | METHODS AND COMPOSITIONS TO PROMOTE AND ENHANCE T-CELL-MEDIATED IMMUNE RESPONSES THROUGH ADCC TARGETING OF CELLS THAT EXPRESS CD39 |
WO2021028752A1 (en) | 2019-08-15 | 2021-02-18 | Janssen Biotech, Inc. | Anti-tfn antibodies for treating type i diabetes |
KR20220053007A (en) | 2019-08-30 | 2022-04-28 | 아게누스 인코포레이티드 | Anti-CD96 antibodies and methods of use thereof |
EP4025303A1 (en) | 2019-09-04 | 2022-07-13 | Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE) | Herv inhibitors for use in treating tauopathies |
KR20220088847A (en) | 2019-09-04 | 2022-06-28 | 주식회사 와이바이오로직스 | Anti-VSIG4 antibodies or antigen-binding fragments and uses thereof |
KR20220057596A (en) | 2019-09-05 | 2022-05-09 | 포세이다 테라퓨틱스, 인크. | Allogeneic Cell Compositions and Methods of Use |
WO2021050645A1 (en) | 2019-09-12 | 2021-03-18 | Genentech, Inc. | Compositions and methods of treating lupus nephritis |
KR20220062500A (en) | 2019-09-16 | 2022-05-17 | 서피스 온콜로지, 인크. | Anti-CD39 Antibody Compositions and Methods |
TW202124446A (en) | 2019-09-18 | 2021-07-01 | 瑞士商諾華公司 | Combination therapies with entpd2 antibodies |
AU2020349509A1 (en) | 2019-09-18 | 2022-03-31 | Genentech, Inc. | Anti-KLK7 antibodies, anti-KLK5 antibodies, multispecific anti-KLK5/KLK7 antibodies, and methods of use |
WO2021053559A1 (en) | 2019-09-18 | 2021-03-25 | Novartis Ag | Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies |
TW202126699A (en) | 2019-09-20 | 2021-07-16 | 美商建南德克公司 | Dosing for anti-tryptase antibodies |
KR20220066346A (en) | 2019-09-25 | 2022-05-24 | 서피스 온콜로지, 인크. | Anti-IL-27 antibodies and uses thereof |
EP4041767A1 (en) | 2019-09-26 | 2022-08-17 | StCube & Co. | Antibodies specific to glycosylated ctla-4 and methods of use thereof |
CN114829401A (en) | 2019-09-27 | 2022-07-29 | 南京金斯瑞生物科技有限公司 | anti-VHH domain antibodies and uses thereof |
WO2021059075A1 (en) | 2019-09-27 | 2021-04-01 | Janssen Biotech, Inc. | Anti-ceacam antibodies and uses thereof |
AU2020351734A1 (en) | 2019-09-27 | 2022-04-14 | Genentech, Inc. | Dosing for treatment with anti-TIGIT and anti-PD-L1 antagonist antibodies |
KR20220088438A (en) | 2019-10-09 | 2022-06-27 | 주식회사 에스티큐브앤컴퍼니 | Antibodies specific for glycosylated LAG3 and methods of use thereof |
EP4045090A1 (en) | 2019-10-18 | 2022-08-24 | Genentech, Inc. | Methods of using anti-cd79b immunoconjugates to treat diffuse large b-cell lymphoma |
AU2020370832A1 (en) | 2019-10-21 | 2022-05-19 | Novartis Ag | TIM-3 inhibitors and uses thereof |
CN114786679A (en) | 2019-10-21 | 2022-07-22 | 诺华股份有限公司 | Combination therapy with Vernetork and TIM-3 inhibitors |
EP3812008A1 (en) | 2019-10-23 | 2021-04-28 | Gamamabs Pharma | Amh-competitive antagonist antibody |
US11459389B2 (en) | 2019-10-24 | 2022-10-04 | Massachusetts Institute Of Technology | Monoclonal antibodies that bind human CD161 |
KR20220092580A (en) | 2019-11-06 | 2022-07-01 | 제넨테크, 인크. | Diagnosis and treatment methods for the treatment of blood cancer |
WO2021099586A1 (en) | 2019-11-20 | 2021-05-27 | Bavarian Nordic A/S | Recombinant mva viruses for intratumoral and/or intravenous administration for treating cancer |
GB201917480D0 (en) | 2019-11-29 | 2020-01-15 | Univ Oxford Innovation Ltd | Antibodies |
AU2020403145A1 (en) | 2019-12-13 | 2022-07-07 | Genentech, Inc. | Anti-Ly6G6D antibodies and methods of use |
AU2020401319A1 (en) | 2019-12-13 | 2022-06-30 | Alector Llc | Anti-MerTK antibodies and methods of use thereof |
KR20220100963A (en) | 2019-12-18 | 2022-07-18 | 에프. 호프만-라 로슈 아게 | Antibodies that bind to HLA-A2/MAGE-A4 |
US20210188971A1 (en) | 2019-12-19 | 2021-06-24 | Quidel Corporation | Monoclonal antibody fusions |
CN115175937A (en) | 2019-12-20 | 2022-10-11 | 诺华股份有限公司 | Combination of anti-TIM-3 antibody MBG453 and anti-TGF-beta antibody NIS793 with or without decitabine or anti-PD-1 antibody, gabapentin, for the treatment of myelofibrosis and myelodysplastic syndrome |
WO2021127505A1 (en) | 2019-12-20 | 2021-06-24 | Poseida Therapeutics, Inc. | Anti-muc1 compositions and methods of use |
GB201919061D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Multi-specific antibody |
GB201919062D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Antibody |
GB201919058D0 (en) | 2019-12-20 | 2020-02-05 | Ucb Biopharma Sprl | Multi-specific antibodies |
BR112022011723A2 (en) | 2019-12-27 | 2022-09-06 | Chugai Pharmaceutical Co Ltd | ANTI-CTLA-4 ANTIBODY AND USE THEREOF |
TW202138388A (en) | 2019-12-30 | 2021-10-16 | 美商西根公司 | Methods of treating cancer with nonfucosylated anti-cd70 antibodies |
WO2021138407A2 (en) | 2020-01-03 | 2021-07-08 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to cd33 and uses thereof |
WO2021142002A1 (en) | 2020-01-06 | 2021-07-15 | Vaccinex, Inc. | Anti-ccr8 antibodies and uses thereof |
CN110818795B (en) | 2020-01-10 | 2020-04-24 | 上海复宏汉霖生物技术股份有限公司 | anti-TIGIT antibodies and methods of use |
AU2021205440A1 (en) | 2020-01-11 | 2022-09-01 | Scholar Rock,Inc. | TGF-beta inhibitors and use thereof |
MX2022008609A (en) | 2020-01-11 | 2022-11-10 | Scholar Rock Inc | Tgfî² inhibitors and use thereof. |
TW202140553A (en) | 2020-01-13 | 2021-11-01 | 美商威特拉公司 | Antibody molecules to c5ar1 and uses thereof |
IL294388A (en) | 2020-01-14 | 2022-08-01 | Synthekine Inc | Il2 orthologs and methods of use |
KR20220128389A (en) | 2020-01-17 | 2022-09-20 | 노파르티스 아게 | A combination comprising a TIM-3 inhibitor and a hypomethylating agent for use in treating myelodysplastic syndrome or chronic myelomonocytic leukemia |
WO2021194481A1 (en) | 2020-03-24 | 2021-09-30 | Genentech, Inc. | Dosing for treatment with anti-tigit and anti-pd-l1 antagonist antibodies |
WO2022050954A1 (en) | 2020-09-04 | 2022-03-10 | Genentech, Inc. | Dosing for treatment with anti-tigit and anti-pd-l1 antagonist antibodies |
CN115397848A (en) | 2020-02-05 | 2022-11-25 | 拉利玛生物医药公司 | TAT peptide binding proteins and uses thereof |
AU2021218927A1 (en) | 2020-02-10 | 2022-09-22 | Shanghai Escugen Biotechnology Co., Ltd. | Claudin 18.2 antibody and use thereof |
EP4105237A4 (en) | 2020-02-10 | 2024-03-27 | Shanghai Escugen Biotechnology Co Ltd | Cldn18.2 antibody and use thereof |
TW202144395A (en) | 2020-02-12 | 2021-12-01 | 日商中外製藥股份有限公司 | Anti-CD137 antigen-binding molecule for use in cancer treatment |
WO2021160269A1 (en) | 2020-02-13 | 2021-08-19 | UCB Biopharma SRL | Anti cd44-ctla4 bispecific antibodies |
EP4103611B1 (en) | 2020-02-13 | 2024-03-27 | UCB Biopharma SRL | Bispecific antibodies binding hvem and cd9 |
EP4103609A1 (en) | 2020-02-13 | 2022-12-21 | UCB Biopharma SRL | Bispecific antibodies against cd9 and cd7 |
US20230151108A1 (en) | 2020-02-13 | 2023-05-18 | UCB Biopharma SRL | Bispecific antibodies against cd9 and cd137 |
US20230151109A1 (en) | 2020-02-13 | 2023-05-18 | UCB Biopharma SRL | Bispecific antibodies against cd9 |
EP4103285A2 (en) | 2020-02-14 | 2022-12-21 | Jounce Therapeutics, Inc. | Antibodies and fusion proteins that bind to ccr8 and uses thereof |
EP4106788A1 (en) | 2020-02-18 | 2022-12-28 | Alector LLC | Pilra antibodies and methods of use thereof |
JP2023516941A (en) | 2020-02-28 | 2023-04-21 | 上海復宏漢霖生物技術股▲フン▼有限公司 | Anti-CD137 constructs, multispecific antibodies and uses thereof |
KR20220148209A (en) | 2020-02-28 | 2022-11-04 | 상하이 헨리우스 바이오테크, 인크. | Anti-CD137 constructs and uses thereof |
KR20220151195A (en) | 2020-03-06 | 2022-11-14 | 오엔에이 테라퓨틱스 에스.엘. | Anti-CD36 antibodies and their use to treat cancer |
IL296241A (en) | 2020-03-10 | 2022-11-01 | Massachusetts Inst Technology | Compositions and methods for immunotherapy of npm1c-positive cancer |
MX2022011289A (en) | 2020-03-11 | 2022-12-08 | Fundacio Inst De Recerca Contra La Leucemia Josep Carreras | Cd22 targeting-moiety for the treatment of b-cell acute lymphoblastic leukemia (b-all). |
EP4118107A1 (en) | 2020-03-11 | 2023-01-18 | Poseida Therapeutics, Inc. | Chimeric stimulatory receptors and methods of use in t cell activation and differentiation |
KR20220152262A (en) | 2020-03-13 | 2022-11-15 | 제넨테크, 인크. | Anti-interleukin-33 antibodies and uses thereof |
AU2021236660A1 (en) | 2020-03-19 | 2022-08-18 | Genentech, Inc. | Isoform-selective anti-TGF-beta antibodies and methods of use |
BR112022018847A2 (en) | 2020-03-24 | 2022-11-22 | Genentech Inc | ANTIBODIES, NUCLEIC ACID, HOST CELL, CONJUGATES, PHARMACEUTICAL COMPOSITION, LONG-ACTION DELIVERY DEVICE FOR OCULAR DELIVERY, METHOD FOR TREAT A DISORDER AND USE OF THE ANTIBODY |
WO2021202473A2 (en) | 2020-03-30 | 2021-10-07 | Danisco Us Inc | Engineered antibodies |
WO2021198034A1 (en) | 2020-03-30 | 2021-10-07 | F. Hoffmann-La Roche Ag | Antibody that binds to vegf and pdgf-b and methods of use |
WO2021202590A1 (en) | 2020-03-31 | 2021-10-07 | Alector Llc | Anti-mertk antibodies and methods of use thereof |
US20230107644A1 (en) | 2020-04-01 | 2023-04-06 | University Of Rochester | Monoclonal antibodies against the hemagglutinin (ha) and neuraminidase (na) of influenza h3n2 viruses |
CA3178465A1 (en) | 2020-04-03 | 2021-10-07 | Visterra, Inc. | Antibody molecule-drug conjugates and uses thereof |
CN115698717A (en) | 2020-04-03 | 2023-02-03 | 基因泰克公司 | Methods of treatment and diagnosis of cancer |
CA3175523A1 (en) | 2020-04-13 | 2021-10-21 | Antti Virtanen | Methods, complexes and kits for detecting or determining an amount of a .beta.-coronavirus antibody in a sample |
CA3174777A1 (en) | 2020-04-14 | 2021-10-21 | Eric M. Ostertag | Compositions and methods for use in the treatment of cancer |
BR112022020629A2 (en) | 2020-04-15 | 2022-11-29 | Hoffmann La Roche | INTERLEUKIN-7 (IL-7) POLYPEPTIDE, IMMUNOCONJUGATE, ONE OR MORE POLYNUCLEOTIDES ISOLATED, HOST CELL, METHODS FOR PRODUCING AN IL-7 POLYPEPTIDE MUTANT OR AN IMMUNOCONJUGATE, FOR TREAT A DISEASE, AND FOR STIMULATING THE IMMUNE SYSTEM, IL-7 POLYPEPTIDE OR IMMUNOCONJUGATE 7 MUTANT, PHARMACEUTICAL COMPOSITION, USE OF THE IL-7 MUTANT POLYPEPTIDE AND INVENTION |
JP2023522930A (en) | 2020-04-24 | 2023-06-01 | ジェネンテック, インコーポレイテッド | Methods of Using Anti-CD79b Immunoconjugates |
WO2021217085A1 (en) | 2020-04-24 | 2021-10-28 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to t cell related cancer cells and uses thereof |
WO2021222167A1 (en) | 2020-04-28 | 2021-11-04 | Genentech, Inc. | Methods and compositions for non-small cell lung cancer immunotherapy |
KR20230002261A (en) | 2020-04-28 | 2023-01-05 | 더 락커펠러 유니버시티 | Anti-SARS-COV-2 Neutralizing Antibodies and Methods of Using The Same |
CR20220598A (en) | 2020-04-30 | 2023-01-17 | Genentech Inc | Kras specific antibodies and uses thereof |
WO2021220218A1 (en) | 2020-05-01 | 2021-11-04 | Novartis Ag | Immunoglobulin variants |
JP2023523794A (en) | 2020-05-01 | 2023-06-07 | ノバルティス アーゲー | engineered immunoglobulin |
CN116963782A (en) | 2020-05-03 | 2023-10-27 | 联宁(苏州)生物制药有限公司 | Antibody drug conjugates comprising anti-TROP-2 antibodies |
WO2021226290A1 (en) | 2020-05-05 | 2021-11-11 | 10X Genomics, Inc. | Methods for identification of antigen-binding molecules |
US20210355196A1 (en) | 2020-05-17 | 2021-11-18 | Astrazeneca Uk Limited | Sars-cov-2 antibodies and methods of selecting and using the same |
WO2021239666A1 (en) | 2020-05-26 | 2021-12-02 | Diaccurate | Therapeutic methods |
WO2021238886A1 (en) | 2020-05-27 | 2021-12-02 | Staidson (Beijing) Biopharmaceuticals Co., Ltd. | Antibodies specifically recognizing nerve growth factor and uses thereof |
CN116529260A (en) | 2020-06-02 | 2023-08-01 | 当康生物技术有限责任公司 | anti-CD 93 constructs and uses thereof |
MX2022015376A (en) | 2020-06-02 | 2023-04-14 | Dynamicure Biotechnology Llc | Anti-cd93 constructs and uses thereof. |
EP4158058A1 (en) | 2020-06-02 | 2023-04-05 | 10X Genomics, Inc. | Enrichment of nucleic acid sequences |
WO2021247908A1 (en) | 2020-06-03 | 2021-12-09 | Bionecure Therapeutics, Inc. | Trophoblast cell-surface antigen-2 (trop-2) antibodies |
JP2023527918A (en) | 2020-06-08 | 2023-06-30 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Anti-HBV antibody and method of use |
GB202008651D0 (en) | 2020-06-09 | 2020-07-22 | Univ Newcastle | Method of identifying complement modulators |
CN115698719A (en) | 2020-06-12 | 2023-02-03 | 基因泰克公司 | Methods and compositions for cancer immunotherapy |
AU2021293038A1 (en) | 2020-06-16 | 2023-02-02 | F. Hoffmann-La Roche Ag | Methods and compositions for treating triple-negative breast cancer |
IL298946A (en) | 2020-06-18 | 2023-02-01 | Genentech Inc | Treatment with anti-tigit antibodies and pd-1 axis binding antagonists |
MX2022016591A (en) | 2020-06-24 | 2023-04-20 | Visterra Inc | Antibody molecules to april and uses thereof. |
WO2022010797A2 (en) | 2020-07-07 | 2022-01-13 | Bionecure Therapeutics, Inc. | Novel maytansinoids as adc payloads and their use for the treatment of cancer |
TW202216779A (en) | 2020-07-17 | 2022-05-01 | 美商輝瑞股份有限公司 | Therapeutic antibodies and their uses |
PE20231300A1 (en) | 2020-07-17 | 2023-08-24 | Genentech Inc | ANTI-NOTCH2 ANTIBODIES AND METHODS OF USE |
CA3188938A1 (en) | 2020-07-20 | 2022-01-27 | Astrazeneca Uk Limited | Sars-cov-2 proteins, anti-sars-cov-2 antibodies, and methods of using the same |
JP2023535409A (en) | 2020-07-21 | 2023-08-17 | ジェネンテック, インコーポレイテッド | Antibody-Conjugated Chemical Inducers of BRM Degradation and Methods of BRM Degradation |
KR20230040331A (en) | 2020-07-21 | 2023-03-22 | 알로젠 테라퓨틱스 인코포레이티드 | Chimeric Antigen Receptors with Enhanced Signal Transduction and Activity and Uses Thereof |
GB2597532A (en) | 2020-07-28 | 2022-02-02 | Femtogenix Ltd | Cytotoxic compounds |
WO2022026592A2 (en) | 2020-07-28 | 2022-02-03 | Celltas Bio, Inc. | Antibody molecules to coronavirus and uses thereof |
WO2022026763A1 (en) | 2020-07-29 | 2022-02-03 | Dynamicure Biotechnology Llc | Anti-cd93 constructs and uses thereof |
EP4193149A1 (en) | 2020-08-04 | 2023-06-14 | Abbott Laboratories | Improved methods and kits for detecting sars-cov-2 protein in a sample |
PE20231187A1 (en) | 2020-08-18 | 2023-08-15 | Cephalon Llc | ANTI-PAR-2 ANTIBODIES AND METHODS OF USE THEREOF |
WO2022046922A2 (en) | 2020-08-26 | 2022-03-03 | Marengo Therapeutics, Inc. | Antibody molecules that bind to nkp30 and uses thereof |
EP4204458A1 (en) | 2020-08-26 | 2023-07-05 | Marengo Therapeutics, Inc. | Methods of detecting trbc1 or trbc2 |
WO2022046920A2 (en) | 2020-08-26 | 2022-03-03 | Marengo Therapeutics, Inc. | Multifunctional molecules that bind to calreticulin and uses thereof |
EP4204021A1 (en) | 2020-08-31 | 2023-07-05 | Advanced Accelerator Applications International S.A. | Method of treating psma-expressing cancers |
US20230338587A1 (en) | 2020-08-31 | 2023-10-26 | Advanced Accelerator Applications International Sa | Method of treating psma-expressing cancers |
WO2022053650A1 (en) | 2020-09-11 | 2022-03-17 | Medimmune Limited | Therapeutic b7-h4 binding molecules |
AU2021342349A1 (en) | 2020-09-12 | 2023-05-25 | Medimmune Limited | A scoring method for an anti-b7h4 antibody-drug conjugate therapy |
EP3981789A1 (en) | 2020-10-12 | 2022-04-13 | Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives | Anti-lilrb antibodies and uses thereof |
EP4228764A1 (en) | 2020-10-14 | 2023-08-23 | Five Prime Therapeutics, Inc. | Anti-c-c chemokine receptor 8 (ccr8) antibodies and methods of use thereof |
JP2023547795A (en) | 2020-10-15 | 2023-11-14 | ユーシービー バイオファルマ エスアールエル | Binding molecules that multimerize CD45 |
US20230382978A1 (en) | 2020-10-15 | 2023-11-30 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Antibody specific for sars-cov-2 receptor binding domain and therapeutic methods |
CA3190782A1 (en) | 2020-10-20 | 2022-04-28 | F. Hoffmann-La Roche Ag | Combination therapy of pd-1 axis binding antagonists and lrrk2 inhibitors |
WO2022087274A1 (en) | 2020-10-21 | 2022-04-28 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Antibodies that neutralize type-i interferon (ifn) activity |
AR124562A1 (en) | 2020-10-28 | 2023-04-12 | Hoffmann La Roche | ENHANCED ANTIGEN BINDING RECEPTORS |
JP2023548064A (en) | 2020-11-04 | 2023-11-15 | ジェネンテック, インコーポレイテッド | Administration for treatment with anti-CD20/anti-CD3 bispecific antibody and anti-CD79B antibody drug conjugate |
US20220162329A1 (en) | 2020-11-04 | 2022-05-26 | Genentech, Inc. | Dosing for treatment with anti-cd20/anti-cd3 bispecific antibodies |
US11919945B2 (en) | 2020-11-04 | 2024-03-05 | The Rockefeller University | Neutralizing anti-SARS-CoV-2 antibodies |
WO2022098628A2 (en) | 2020-11-04 | 2022-05-12 | Genentech, Inc. | Subcutaneous dosing of anti-cd20/anti-cd3 bispecific antibodies |
EP4251647A1 (en) | 2020-11-24 | 2023-10-04 | Bio-Techne Corporation | Anti-severe acute respiratory syndrome coronavirus antibodies |
WO2023102384A1 (en) | 2021-11-30 | 2023-06-08 | Abbott Laboratories | Use of one or more biomarkers to determine traumatic brain injury (tbi) in a subject having received a head computerized tomography scan that is negative for a tbi |
MX2023006426A (en) | 2020-12-01 | 2023-07-17 | Aptevo Res & Development Llc | Heterodimeric psma and cd3-binding bispecific antibodies. |
WO2022119841A1 (en) | 2020-12-01 | 2022-06-09 | Abbott Laboratories | Use of one or more biomarkers to determine traumatic brain injury (tbi) in a subject having received a head computerized tomography scan that is negative for a tbi |
US20220168293A1 (en) | 2020-12-02 | 2022-06-02 | Pfizer Inc. | Time to resolution of axitinib-related adverse events |
AU2021392039A1 (en) | 2020-12-02 | 2023-06-29 | Alector Llc | Methods of use of anti-sortilin antibodies |
MX2023006599A (en) | 2020-12-04 | 2023-06-19 | Visterra Inc | Methods of using interleukin-2 agents. |
KR20230118128A (en) | 2020-12-07 | 2023-08-10 | 유씨비 바이오파마 에스알엘 | Antibodies to Interleukin-22 |
AU2021395729A1 (en) | 2020-12-07 | 2023-07-13 | UCB Biopharma SRL | Multi-specific antibodies and antibody combinations |
CR20230263A (en) | 2020-12-17 | 2023-08-21 | Hoffmann La Roche | Anti-hla-g antibodies and use thereof |
WO2022132904A1 (en) | 2020-12-17 | 2022-06-23 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies targeting sars-cov-2 |
KR20230132470A (en) | 2020-12-18 | 2023-09-15 | 키닉사 파마슈티컬스, 리미티드 | Protein compositions and methods of producing and using them |
EP4271998A1 (en) | 2020-12-30 | 2023-11-08 | Abbott Laboratories | Methods for determining sars-cov-2 antigen and anti-sars-cov-2 antibody in a sample |
EP4271482A2 (en) | 2020-12-31 | 2023-11-08 | Alamar Biosciences, Inc. | Binder molecules with high affinity and/ or specificity and methods of making and use thereof |
WO2022148853A1 (en) | 2021-01-11 | 2022-07-14 | F. Hoffmann-La Roche Ag | Immunoconjugates |
AU2022207708A1 (en) | 2021-01-13 | 2023-07-27 | Daiichi Sankyo Company, Limited | Antibody-pyrrolobenzodiazepine derivative conjugate |
CN117425500A (en) | 2021-01-13 | 2024-01-19 | 纪念斯隆凯特琳癌症中心 | anti-DLL 3 antibody-drug conjugates |
US20220227844A1 (en) | 2021-01-15 | 2022-07-21 | The Rockefeller University | Neutralizing anti-sars-cov-2 antibodies |
TW202245825A (en) | 2021-01-20 | 2022-12-01 | 美商威特拉公司 | Interleukin-2 agents and uses thereof |
JP2024505049A (en) | 2021-01-29 | 2024-02-02 | ノバルティス アーゲー | Administration modes for anti-CD73 and anti-ENTPD2 antibodies and their uses |
AU2022220611A1 (en) | 2021-02-09 | 2023-08-24 | University Of Georgia Research Foundation, Inc. | Human monoclonal antibodies against pneumococcal antigens |
EP4291226A1 (en) | 2021-02-09 | 2023-12-20 | The United States of America, as represented by the Secretary, Department of Health and Human Services | Antibodies targeting the spike protein of coronaviruses |
WO2022182872A2 (en) | 2021-02-24 | 2022-09-01 | Alladapt Immunotherapeutics, Inc. | Compositions and methods for identification of cross-reactive allergenic proteins and treatment of allergies |
EP4301362A1 (en) | 2021-03-02 | 2024-01-10 | CGRP Diagnostics GmbH | Treatment and/or reduction of occurrence of migraine |
CN117440832A (en) | 2021-03-03 | 2024-01-23 | 索伦托药业有限公司 | Antibody-drug conjugates comprising anti-BCMA antibodies |
US20240158503A1 (en) | 2021-03-03 | 2024-05-16 | Pierre Fabre Medicament | Anti-vsig4 antibody or antigen binding fragment and uses thereof |
WO2022187440A1 (en) | 2021-03-03 | 2022-09-09 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | La protien as a novel regulator of osteoclastogenesis |
US20240141048A1 (en) | 2021-03-05 | 2024-05-02 | Dynamicure Biotechnology Llc | Anti-vista constructs and uses thereof |
JP2024510588A (en) | 2021-03-12 | 2024-03-08 | ヤンセン バイオテツク,インコーポレーテツド | Method of treating psoriatic arthritis patients with inadequate response to TNF therapy with anti-IL23-specific antibodies |
JP2024512377A (en) | 2021-03-12 | 2024-03-19 | ジェネンテック, インコーポレイテッド | Anti-KLK7 antibodies, anti-KLK5 antibodies, multispecific anti-KLK5/KLK7 antibodies, and methods of use |
US20220298236A1 (en) | 2021-03-12 | 2022-09-22 | Janssen Biotech, Inc. | Safe and Effective Method of Treating Psoriatic Arthritis with Anti-IL23 Specific Antibody |
CA3213599A1 (en) | 2021-03-15 | 2022-09-22 | Genentech, Inc. | Compositions and methods of treating lupus nephritis |
MX2023010917A (en) | 2021-03-18 | 2023-09-27 | Medimmune Ltd | Therapeutic binding molecule that binds to ccr9. |
WO2022195551A1 (en) | 2021-03-18 | 2022-09-22 | Novartis Ag | Biomarkers for cancer and methods of use thereof |
CN116981696A (en) | 2021-03-18 | 2023-10-31 | 艾莱克特有限责任公司 | anti-TMEM 106B antibodies and methods of use thereof |
WO2022197877A1 (en) | 2021-03-19 | 2022-09-22 | Genentech, Inc. | Methods and compositions for time delayed bio-orthogonal release of cytotoxic agents |
WO2022204274A1 (en) | 2021-03-23 | 2022-09-29 | Alector Llc | Anti-tmem106b antibodies for treating and preventing coronavirus infections |
CN117616041A (en) | 2021-03-25 | 2024-02-27 | 当康生物技术有限责任公司 | anti-IGFBP 7 constructs and uses thereof |
WO2022204581A2 (en) | 2021-03-26 | 2022-09-29 | Scholar Rock, Inc. | Tgf-beta inhibitors and use thereof |
EP4067381A1 (en) | 2021-04-01 | 2022-10-05 | Julius-Maximilians-Universität Würzburg | Novel tnfr2 binding molecules |
TW202304979A (en) | 2021-04-07 | 2023-02-01 | 瑞士商諾華公司 | USES OF ANTI-TGFβ ANTIBODIES AND OTHER THERAPEUTIC AGENTS FOR THE TREATMENT OF PROLIFERATIVE DISEASES |
KR20240004462A (en) | 2021-04-08 | 2024-01-11 | 마렝고 테라퓨틱스, 인크. | Multifunctional molecules that bind to TCR and their uses |
AR125344A1 (en) | 2021-04-15 | 2023-07-05 | Chugai Pharmaceutical Co Ltd | ANTI-C1S ANTIBODY |
IL307821A (en) | 2021-04-30 | 2023-12-01 | Hoffmann La Roche | Dosing for treatment with anti-cd20/anti-cd3 bispecific antibody |
JP2024517535A (en) | 2021-04-30 | 2024-04-23 | エフ・ホフマン-ラ・ロシュ・アクチェンゲゼルシャフト | Administration of combination therapy with anti-CD20/anti-CD3 bispecific antibody and anti-CD79B antibody drug conjugate |
AR125732A1 (en) | 2021-05-03 | 2023-08-09 | UCB Biopharma SRL | ANTI-TREM1 ANTIBODIES |
EP4334343A2 (en) | 2021-05-06 | 2024-03-13 | The Rockefeller University | Neutralizing anti-sars- cov-2 antibodies and methods of use thereof |
CA3215856A1 (en) | 2021-05-07 | 2022-11-10 | Alison O'neill | Anti-il-27 antibodies and uses thereof |
IL308351A (en) | 2021-05-12 | 2024-01-01 | Genentech Inc | Methods of using anti-cd79b immunoconjugates to treat diffuse large b-cell lymphoma |
WO2022245920A1 (en) | 2021-05-18 | 2022-11-24 | Abbott Laboratories | Methods of evaluating brain injury in a pediatric subject |
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 |
JP2024520497A (en) | 2021-05-28 | 2024-05-24 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | Methods for detecting CM-TMA biomarkers |
WO2022256313A1 (en) | 2021-06-01 | 2022-12-08 | 10X Genomics, Inc. | Validation of a unique molecular identifier associated with a nucleic acid sequence of interest |
WO2022256723A2 (en) | 2021-06-03 | 2022-12-08 | Scholar Rock, Inc. | Tgf-beta inhibitors and therapeutic use thereof |
EP4155321A1 (en) | 2021-06-04 | 2023-03-29 | Chugai Seiyaku Kabushiki Kaisha | Anti-ddr2 antibodies and uses thereof |
EP4351595A1 (en) | 2021-06-07 | 2024-04-17 | Providence Health & Services - Oregon | Cxcr5, pd-1, and icos expressing tumor reactive cd4 t cells and their use |
WO2022261183A2 (en) | 2021-06-08 | 2022-12-15 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating and/or identifying an agent for treating intestinal cancers |
EP4351582A1 (en) | 2021-06-09 | 2024-04-17 | F. Hoffmann-La Roche AG | Combination of a particular braf inhibitor (paradox breaker) and a pd-1 axis binding antagonist for use in the treatment of cancer |
IL309349A (en) | 2021-06-14 | 2024-02-01 | argenx BV | Anti-il-9 antibodies and methods of use thereof |
BR112023026199A2 (en) | 2021-06-14 | 2024-03-05 | Abbott Lab | METHODS FOR DIAGNOSING OR ASSISTING IN THE DIAGNOSIS OF BRAIN INJURY CAUSED BY ACOUSTIC ENERGY, ELECTROMAGNETIC ENERGY, OVERPRESSURIZATION WAVE AND/OR GUST OF WIND |
WO2022266223A1 (en) | 2021-06-16 | 2022-12-22 | Alector Llc | Bispecific anti-mertk and anti-pdl1 antibodies and methods of use thereof |
EP4355783A1 (en) | 2021-06-16 | 2024-04-24 | Alector LLC | Monovalent anti-mertk antibodies and methods of use thereof |
WO2022266660A1 (en) | 2021-06-17 | 2022-12-22 | Amberstone Biosciences, Inc. | Anti-cd3 constructs and uses thereof |
AU2022299185A1 (en) | 2021-06-23 | 2024-01-25 | Scholar Rock, Inc. | A myostatin pathway inhibitor in combination with a glp-1 pathway activator for use in treating metabolic disorders |
IL308633A (en) | 2021-06-25 | 2024-01-01 | Chugai Pharmaceutical Co Ltd | Use of anti-ctla-4 antibody |
IL309115A (en) | 2021-06-25 | 2024-02-01 | Chugai Pharmaceutical Co Ltd | Anti–ctla-4 antibody |
EP4362977A1 (en) | 2021-06-29 | 2024-05-08 | Seagen Inc. | Methods of treating cancer with a combination of a nonfucosylated anti-cd70 antibody and a cd47 antagonist |
EP4367139A1 (en) | 2021-07-08 | 2024-05-15 | Staidson Biopharma Inc. | Antibodies specifically recognizing tnfr2 and uses thereof |
US20230040065A1 (en) | 2021-07-09 | 2023-02-09 | Janssen Biotech, Inc. | Manufacturing Methods for Producing Anti-TNF Antibody Compositions |
WO2023281466A1 (en) | 2021-07-09 | 2023-01-12 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
CN117957251A (en) | 2021-07-09 | 2024-04-30 | 詹森生物科技公司 | Method for preparing anti-TNF antibody compositions |
WO2023285878A1 (en) | 2021-07-13 | 2023-01-19 | Aviation-Ophthalmology | Methods for detecting, treating, and preventing gpr68-mediated ocular diseases, disorders, and conditions |
KR20240058075A (en) | 2021-07-14 | 2024-05-03 | 스테이드슨 (베이징) 바이오팔마슈티칼스 캄퍼니 리미티드 | Antibodies specifically identifying CD40 and their applications |
KR20240034234A (en) | 2021-07-14 | 2024-03-13 | 2세븐티 바이오, 인코포레이티드 | Engineered T cell receptor fused to binding domain from antibody |
WO2023004386A1 (en) | 2021-07-22 | 2023-01-26 | Genentech, Inc. | Brain targeting compositions and methods of use thereof |
WO2023001884A1 (en) | 2021-07-22 | 2023-01-26 | F. Hoffmann-La Roche Ag | Heterodimeric fc domain antibodies |
KR20240042476A (en) | 2021-07-30 | 2024-04-02 | 오엔에이 테라퓨틱스 에스.엘. | Anti-CD36 antibodies and their use to treat cancer |
CN117794953A (en) | 2021-08-03 | 2024-03-29 | 豪夫迈·罗氏有限公司 | Bispecific antibodies and methods of use |
US11807685B2 (en) | 2021-08-05 | 2023-11-07 | The Uab Research Foundation | Anti-CD47 antibody and uses thereof |
WO2023012343A1 (en) | 2021-08-06 | 2023-02-09 | Institut Du Cancer De Montpellier | Methods for the treatment of cancer |
AU2022328390A1 (en) | 2021-08-10 | 2024-03-21 | Adimab, Llc | Anti-gdf15 antibodies, compositions and uses thereof |
WO2023019239A1 (en) | 2021-08-13 | 2023-02-16 | Genentech, Inc. | Dosing for anti-tryptase antibodies |
GB202111905D0 (en) | 2021-08-19 | 2021-10-06 | UCB Biopharma SRL | Antibodies |
WO2023026205A1 (en) | 2021-08-24 | 2023-03-02 | Cgrp Diagnostics Gmbh | Preventative treatment of migraine |
WO2023025927A1 (en) | 2021-08-26 | 2023-03-02 | Glycanostics S.R.O | Glycoprotein biomarkers for diagnosing cancer |
WO2023034750A1 (en) | 2021-08-30 | 2023-03-09 | Genentech, Inc. | Anti-polyubiquitin multispecific antibodies |
WO2023032955A1 (en) | 2021-08-31 | 2023-03-09 | 大正製薬株式会社 | Anti-growth hormone antibody |
WO2023034777A1 (en) | 2021-08-31 | 2023-03-09 | Abbott Laboratories | Methods and systems of diagnosing brain injury |
CN113603775B (en) | 2021-09-03 | 2022-05-20 | 江苏荃信生物医药股份有限公司 | Anti-human interleukin-33 monoclonal antibody and application thereof |
AR126971A1 (en) | 2021-09-03 | 2023-12-06 | Univ Of Bern | COMPOSITIONS AND METHODS FOR TREATING LONG QT SYNDROME |
CN113683694B (en) | 2021-09-03 | 2022-05-13 | 江苏荃信生物医药股份有限公司 | Anti-human TSLP monoclonal antibody and application thereof |
CA3231890A1 (en) | 2021-09-14 | 2023-03-23 | Jan Tkac | Use of lectins to determine mammaglobin-a glycoforms in breast cancer |
AU2022345881A1 (en) | 2021-09-20 | 2024-03-21 | Alnylam Pharmaceuticals, Inc. | Inhibin subunit beta e (inhbe) modulator compositions and methods of use thereof |
WO2023044483A2 (en) | 2021-09-20 | 2023-03-23 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
CA3232176A1 (en) | 2021-09-30 | 2023-04-06 | Beth MCQUISTON | Methods and systems of diagnosing brain injury |
WO2023056069A1 (en) | 2021-09-30 | 2023-04-06 | Angiex, Inc. | Degrader-antibody conjugates and methods of using same |
WO2023056403A1 (en) | 2021-09-30 | 2023-04-06 | Genentech, Inc. | Methods for treatment of hematologic cancers using anti-tigit antibodies, anti-cd38 antibodies, and pd-1 axis binding antagonists |
AU2022360244A1 (en) | 2021-10-04 | 2024-04-11 | Poseida Therapeutics, Inc. | Transposon compositions and methods of use thereof |
WO2023057882A1 (en) | 2021-10-05 | 2023-04-13 | Pfizer Inc. | Combinations of azalactam compounds with a pd-1 axis binding antagonist for the treatment of cancer |
JPWO2023058723A1 (en) | 2021-10-08 | 2023-04-13 | ||
CN118139648A (en) | 2021-10-14 | 2024-06-04 | 豪夫迈·罗氏有限公司 | Substituted PD1-IL7v immunoconjugates for the treatment of cancer |
AU2022362681A1 (en) | 2021-10-14 | 2024-04-04 | F. Hoffmann-La Roche Ag | New interleukin-7 immunoconjugates |
CA3235627A1 (en) | 2021-10-18 | 2023-04-27 | Byomass Inc. | Anti-activin a antibodies, compositions and uses thereof |
WO2023069919A1 (en) | 2021-10-19 | 2023-04-27 | Alector Llc | Anti-cd300lb antibodies and methods of use thereof |
WO2023073615A1 (en) | 2021-10-29 | 2023-05-04 | Janssen Biotech, Inc. | Methods of treating crohn's disease with anti-il23 specific antibody |
EP4177266A1 (en) | 2021-11-05 | 2023-05-10 | Katholieke Universiteit Leuven | Neutralizing anti-sars-cov-2 human antibodies |
WO2023086807A1 (en) | 2021-11-10 | 2023-05-19 | Genentech, Inc. | Anti-interleukin-33 antibodies and uses thereof |
WO2023086824A1 (en) | 2021-11-10 | 2023-05-19 | 10X Genomics, Inc. | Methods for identification of antigen-binding molecules |
US20230151087A1 (en) | 2021-11-15 | 2023-05-18 | Janssen Biotech, Inc. | Methods of Treating Crohn's Disease with Anti-IL23 Specific Antibody |
AU2022389969A1 (en) | 2021-11-16 | 2024-05-02 | Genentech, Inc. | Methods and compositions for treating systemic lupus erythematosus (sle) with mosunetuzumab |
WO2023092004A1 (en) | 2021-11-17 | 2023-05-25 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of tau-related disorders |
WO2023095000A1 (en) | 2021-11-23 | 2023-06-01 | Janssen Biotech, Inc. | Method of treating ulcerative colitis with anti-il23 specific antibody |
US20230348614A1 (en) | 2021-11-24 | 2023-11-02 | Visterra, Inc. | Engineered antibody molecules to cd138 and uses thereof |
WO2023097119A2 (en) | 2021-11-29 | 2023-06-01 | Dana-Farber Cancer Institute, Inc. | Methods and compositions to modulate riok2 |
CA3238574A1 (en) | 2021-12-01 | 2023-06-08 | Gregory Babcock | Methods of using interleukin-2 agents |
CA3240822A1 (en) | 2021-12-17 | 2023-06-22 | Tony Lee | Systems and methods for determining uch-l1, gfap, and other biomarkers in blood samples |
CA3240565A1 (en) | 2021-12-17 | 2023-06-22 | Wenfeng Xu | Anti-ox40 antibodies and methods of use |
CA3240585A1 (en) | 2021-12-17 | 2023-06-22 | Wenfeng Xu | Anti-ox40 antibodies, multispecific antibodies and methods of use |
WO2023122213A1 (en) | 2021-12-22 | 2023-06-29 | Byomass Inc. | Targeting gdf15-gfral pathway cross-reference to related applications |
WO2023118508A1 (en) | 2021-12-23 | 2023-06-29 | Bavarian Nordic A/S | Recombinant mva viruses for intraperitoneal administration for treating cancer |
WO2023129942A1 (en) | 2021-12-28 | 2023-07-06 | Abbott Laboratories | Use of biomarkers to determine sub-acute traumatic brain injury (tbi) in a subject having received a head computerized tomography (ct) scan that is negative for a tbi or no head ct scan |
TW202340251A (en) | 2022-01-19 | 2023-10-16 | 美商建南德克公司 | Anti-notch2 antibodies and conjugates and methods of use |
WO2023141576A1 (en) | 2022-01-21 | 2023-07-27 | Poseida Therapeutics, Inc. | Compositions and methods for delivery of nucleic acids |
WO2023147399A1 (en) | 2022-01-27 | 2023-08-03 | The Rockefeller University | Broadly neutralizing anti-sars-cov-2 antibodies targeting the n-terminal domain of the spike protein and methods of use thereof |
WO2023147107A1 (en) | 2022-01-31 | 2023-08-03 | Byomass Inc. | Myeloproliferative conditions |
WO2023150652A1 (en) | 2022-02-04 | 2023-08-10 | Abbott Laboratories | Lateral flow methods, assays, and devices for detecting the presence or measuring the amount of ubiquitin carboxy-terminal hydrolase l1 and/or glial fibrillary acidic protein in a sample |
WO2023150778A1 (en) | 2022-02-07 | 2023-08-10 | Visterra, Inc. | Anti-idiotype antibody molecules and uses thereof |
WO2023154824A1 (en) | 2022-02-10 | 2023-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies that broadly target coronaviruses |
TW202348252A (en) | 2022-02-16 | 2023-12-16 | 英商梅迪繆思有限公司 | Combination therapies for treatment of cancer with therapeutic binding molecules |
WO2023169896A1 (en) | 2022-03-09 | 2023-09-14 | Astrazeneca Ab | BINDING MOLECULES AGAINST FRα |
WO2023170216A1 (en) | 2022-03-11 | 2023-09-14 | Astrazeneca Ab | A SCORING METHOD FOR AN ANTI-FRα ANTIBODY-DRUG CONJUGATE THERAPY |
WO2023175614A1 (en) | 2022-03-15 | 2023-09-21 | Yeda Research And Development Co. Ltd. | Anti glucocorticoid-induced tnfr-related (gitr) protein antibodies and uses thereof |
US20230414750A1 (en) | 2022-03-23 | 2023-12-28 | Hoffmann-La Roche Inc. | Combination treatment of an anti-cd20/anti-cd3 bispecific antibody and chemotherapy |
WO2023180511A1 (en) | 2022-03-25 | 2023-09-28 | F. Hoffmann-La Roche Ag | Improved chimeric receptors |
US20230312703A1 (en) | 2022-03-30 | 2023-10-05 | Janssen Biotech, Inc. | Method of Treating Psoriasis with IL-23 Specific Antibody |
WO2023192436A1 (en) | 2022-03-31 | 2023-10-05 | Alexion Pharmaceuticals, Inc. | Singleplex or multiplexed assay for complement markers in fresh biological samples |
WO2023192478A1 (en) | 2022-04-01 | 2023-10-05 | Bristol-Myers Squibb Company | Combination therapy with anti-il-8 antibodies and anti-pd-1 antibodies for treating cancer |
WO2023196866A1 (en) | 2022-04-06 | 2023-10-12 | Mirobio Limited | Engineered cd200r antibodies and uses thereof |
GB202205203D0 (en) | 2022-04-08 | 2022-05-25 | UCB Biopharma SRL | Combination with inhibitor |
GB202205200D0 (en) | 2022-04-08 | 2022-05-25 | Ucb Biopharma Sprl | Combination with chemotherapy |
WO2023198727A1 (en) | 2022-04-13 | 2023-10-19 | F. Hoffmann-La Roche Ag | Pharmaceutical compositions of anti-cd20/anti-cd3 bispecific antibodies and methods of use |
TW202400637A (en) | 2022-04-25 | 2024-01-01 | 美商威特拉公司 | Antibody molecules to april and uses thereof |
WO2023209177A1 (en) | 2022-04-29 | 2023-11-02 | Astrazeneca Uk Limited | Sars-cov-2 antibodies and methods of using the same |
TW202406934A (en) | 2022-05-03 | 2024-02-16 | 美商建南德克公司 | Anti-ly6e antibodies, immunoconjugates, and uses thereof |
WO2023220695A2 (en) | 2022-05-13 | 2023-11-16 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of her2 positive cancer |
US20230374122A1 (en) | 2022-05-18 | 2023-11-23 | Janssen Biotech, Inc. | Method for Evaluating and Treating Psoriatic Arthritis with IL23 Antibody |
WO2023235699A1 (en) | 2022-05-31 | 2023-12-07 | Jounce Therapeutics, Inc. | Antibodies to lilrb4 and uses thereof |
WO2023240058A2 (en) | 2022-06-07 | 2023-12-14 | Genentech, Inc. | Prognostic and therapeutic methods for cancer |
WO2023240124A1 (en) | 2022-06-07 | 2023-12-14 | Regeneron Pharmaceuticals, Inc. | Pseudotyped viral particles for targeting tcr-expressing cells |
WO2023239803A1 (en) | 2022-06-08 | 2023-12-14 | Angiex, Inc. | Anti-tm4sf1 antibody-drug conjugates comprising cleavable linkers and methods of using same |
WO2023250402A2 (en) | 2022-06-22 | 2023-12-28 | Antlera Therapeutics Inc. | Tetravalent fzd and wnt co-receptor binding antibody molecules and uses thereof |
EP4296279A1 (en) | 2022-06-23 | 2023-12-27 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Anti-transthyretin (ttr) binding proteins and uses thereof |
WO2024006876A1 (en) | 2022-06-29 | 2024-01-04 | Abbott Laboratories | Magnetic point-of-care systems and assays for determining gfap in biological samples |
WO2024015953A1 (en) | 2022-07-15 | 2024-01-18 | Danisco Us Inc. | Methods for producing monoclonal antibodies |
WO2024013727A1 (en) | 2022-07-15 | 2024-01-18 | Janssen Biotech, Inc. | Material and methods for improved bioengineered pairing of antigen-binding variable regions |
WO2024020407A1 (en) | 2022-07-19 | 2024-01-25 | Staidson Biopharma Inc. | Antibodies specifically recognizing b- and t-lymphocyte attenuator (btla) and uses thereof |
WO2024020564A1 (en) | 2022-07-22 | 2024-01-25 | Genentech, Inc. | Anti-steap1 antigen-binding molecules and uses thereof |
WO2024026471A1 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Cd98hc antigen-binding domains and uses therefor |
WO2024026447A1 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Anti-gpnmb antibodies and methods of use thereof |
WO2024026472A2 (en) | 2022-07-29 | 2024-02-01 | Alector Llc | Transferrin receptor antigen-binding domains and uses therefor |
WO2024030829A1 (en) | 2022-08-01 | 2024-02-08 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Monoclonal antibodies that bind to the underside of influenza viral neuraminidase |
WO2024030976A2 (en) | 2022-08-03 | 2024-02-08 | Voyager Therapeutics, Inc. | Compositions and methods for crossing the blood brain barrier |
WO2024050354A1 (en) | 2022-08-31 | 2024-03-07 | Washington University | Alphavirus antigen binding antibodies and uses thereof |
WO2024049949A1 (en) | 2022-09-01 | 2024-03-07 | Genentech, Inc. | Therapeutic and diagnostic methods for bladder cancer |
WO2024050524A1 (en) | 2022-09-01 | 2024-03-07 | University Of Georgia Research Foundation, Inc. | Compositions and methods for directing apolipoprotein l1 to induce mammalian cell death |
WO2024050526A1 (en) | 2022-09-02 | 2024-03-07 | Biomarin Pharmaceutical Inc. | Compositions and methods for treating long qt syndrome |
WO2024054436A1 (en) | 2022-09-06 | 2024-03-14 | Alexion Pharmaceuticals, Inc. | Diagnostic and prognostic biomarker profiles in patients with hematopoietic stem cell transplant-associated thrombotic microangiopathy (hsct-tma) |
WO2024054822A1 (en) | 2022-09-07 | 2024-03-14 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Engineered sars-cov-2 antibodies with increased neutralization breadth |
WO2024054929A1 (en) | 2022-09-07 | 2024-03-14 | Dynamicure Biotechnology Llc | Anti-vista constructs and uses thereof |
WO2024059708A1 (en) | 2022-09-15 | 2024-03-21 | Abbott Laboratories | Biomarkers and methods for differentiating between mild and supermild traumatic brain injury |
WO2024062038A1 (en) | 2022-09-21 | 2024-03-28 | Elthera Ag | Novel binding molecules binding to l1cam |
WO2024086796A1 (en) | 2022-10-20 | 2024-04-25 | Alector Llc | Anti-ms4a4a antibodies with amyloid-beta therapies |
WO2024089551A1 (en) | 2022-10-25 | 2024-05-02 | Janssen Biotech, Inc. | Msln and cd3 binding agents and methods of use thereof |
WO2024097741A1 (en) | 2022-11-04 | 2024-05-10 | Gilead Sciences, Inc. | Anticancer therapies using anti-ccr8 antibody, chemo and immunotherapy combinations |
WO2024102734A1 (en) | 2022-11-08 | 2024-05-16 | Genentech, Inc. | Compositions and methods of treating childhood onset idiopathic nephrotic syndrome |
WO2024100170A1 (en) | 2022-11-11 | 2024-05-16 | F. Hoffmann-La Roche Ag | Antibodies binding to hla-a*02/foxp3 |
US20240199734A1 (en) | 2022-11-22 | 2024-06-20 | Janssen Biotech, Inc. | Method of Treating Ulcerative Colitis with Anti-IL23 Specific Antibody |
Family Cites Families (47)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3853832A (en) * | 1971-04-27 | 1974-12-10 | Harmone Res Foundation | Synthetic human pituitary growth hormone and method of producing it |
US3853833A (en) * | 1971-04-27 | 1974-12-10 | Hormone Res Foundation | Synthetic human growth-promoting and lactogenic hormones and method of producing same |
US4880910A (en) * | 1981-09-18 | 1989-11-14 | Genentech, Inc. | Terminal methionyl bovine growth hormone and its use |
US4593002A (en) * | 1982-01-11 | 1986-06-03 | Salk Institute Biotechnology/Industrial Associates, Inc. | Viruses with recombinant surface proteins |
US4446235A (en) * | 1982-03-22 | 1984-05-01 | Genentech, Inc. | Method for cloning human growth hormone varient genes |
US4670393A (en) * | 1982-03-22 | 1987-06-02 | Genentech, Inc. | DNA vectors encoding a novel human growth hormone-variant protein |
US4673641A (en) * | 1982-12-16 | 1987-06-16 | Molecular Genetics Research And Development Limited Partnership | Co-aggregate purification of proteins |
US4699897A (en) * | 1983-06-04 | 1987-10-13 | Amgen | Biologically active peptides structurally related to regions within growth hormones |
US4888286A (en) * | 1984-02-06 | 1989-12-19 | Creative Biomolecules, Inc. | Production of gene and protein analogs through synthetic gene design using double stranded synthetic oligonucleotides |
US4655160A (en) * | 1985-09-10 | 1987-04-07 | David R. Ligh | Deck box |
US5013653A (en) * | 1987-03-20 | 1991-05-07 | Creative Biomolecules, Inc. | Product and process for introduction of a hinge region into a fusion protein to facilitate cleavage |
IT1223577B (en) * | 1987-12-22 | 1990-09-19 | Eniricerche Spa | IMPROVED PROCEDURE FOR THE PREPARATION OF THE NATURAL HUMAN GROWTH HORMONE IN PURE FORM |
US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
DE68927933T2 (en) * | 1988-09-02 | 1997-08-14 | Dyax Corp | PRODUCTION AND SELECTION OF RECOMBINANT PROTEINS WITH DIFFERENT BINDING POINTS |
US5663143A (en) * | 1988-09-02 | 1997-09-02 | Dyax Corp. | Engineered human-derived kunitz domains that inhibit human neutrophil elastase |
US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
CA2345497A1 (en) * | 1988-10-28 | 1990-04-28 | Genentech, Inc. | Growth hormone variants and method for forming growth hormone variants |
US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5747334A (en) * | 1990-02-15 | 1998-05-05 | The University Of North Carolina At Chapel Hill | Random peptide library |
US5498538A (en) * | 1990-02-15 | 1996-03-12 | The University Of North Carolina At Chapel Hill | Totally synthetic affinity reagents |
US5427908A (en) * | 1990-05-01 | 1995-06-27 | Affymax Technologies N.V. | Recombinant library screening methods |
US5723286A (en) * | 1990-06-20 | 1998-03-03 | Affymax Technologies N.V. | Peptide library and screening systems |
GB9015198D0 (en) * | 1990-07-10 | 1990-08-29 | Brien Caroline J O | Binding substance |
US5770434A (en) * | 1990-09-28 | 1998-06-23 | Ixsys Incorporated | Soluble peptides having constrained, secondary conformation in solution and method of making same |
US5780279A (en) * | 1990-12-03 | 1998-07-14 | Genentech, Inc. | Method of selection of proteolytic cleavage sites by directed evolution and phagemid display |
ATE164395T1 (en) * | 1990-12-03 | 1998-04-15 | Genentech Inc | METHOD FOR ENRICHMENT OF PROTEIN VARIANTS WITH MODIFIED BINDING PROPERTIES |
WO1992018619A1 (en) * | 1991-04-10 | 1992-10-29 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5955341A (en) * | 1991-04-10 | 1999-09-21 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
US5733743A (en) * | 1992-03-24 | 1998-03-31 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1994005781A1 (en) * | 1992-09-04 | 1994-03-17 | The Scripps Research Institute | Phagemids coexpressing a surface receptor and a surface heterologous protein |
CA2115811A1 (en) * | 1993-02-17 | 1994-08-18 | Claus Krebber | A method for in vivo selection of ligand-binding proteins |
SE9304060D0 (en) * | 1993-12-06 | 1993-12-06 | Bioinvent Int Ab | Methods to select specific bacteriophages |
US5811093A (en) * | 1994-04-05 | 1998-09-22 | Exponential Biotherapies, Inc. | Bacteriophage genotypically modified to delay inactivations by the host defense system |
AU7104294A (en) * | 1994-06-10 | 1996-01-05 | Symbiotech, Inc. | Method of detecting compounds utilizing genetically modified lambdoid bacteriophage |
US5516637A (en) * | 1994-06-10 | 1996-05-14 | Dade International Inc. | Method involving display of protein binding pairs on the surface of bacterial pili and bacteriophage |
US5627024A (en) * | 1994-08-05 | 1997-05-06 | The Scripps Research Institute | Lambdoid bacteriophage vectors for expression and display of foreign proteins |
GB9500851D0 (en) * | 1995-01-17 | 1995-03-08 | Bionvent International Ab | Method of selecting specific bacteriophages |
US5702892A (en) * | 1995-05-09 | 1997-12-30 | The United States Of America As Represented By The Department Of Health And Human Services | Phage-display of immunoglobulin heavy chain libraries |
CN1196094A (en) * | 1995-09-07 | 1998-10-14 | 诺沃挪第克公司 | Phage display for detergent enzyme activity |
US5622699A (en) * | 1995-09-11 | 1997-04-22 | La Jolla Cancer Research Foundation | Method of identifying molecules that home to a selected organ in vivo |
WO1997035196A1 (en) * | 1996-03-20 | 1997-09-25 | Dyax Corp. | Engineering affinity ligands for macromolecules |
US6190856B1 (en) * | 1996-05-22 | 2001-02-20 | The Johns Hopkins University | Methods of detection utilizing modified bacteriophage |
JP2000512981A (en) * | 1996-06-06 | 2000-10-03 | ラ ホヤ ファーマシューティカル カンパニー | aPL immunoreactive peptide, conjugate thereof and method of treatment for aPL antibody-mediated pathology |
EP0959894B1 (en) * | 1996-06-10 | 2004-10-13 | The Scripps Research Institute | Use of substrate subtraction libraries to distinguish enzyme specificities |
AU3737297A (en) * | 1996-08-05 | 1998-02-25 | Brigham And Women's Hospital | Bacteriophage-mediated gene therapy |
DE69718341T2 (en) * | 1996-10-08 | 2003-10-30 | Bisys B V U | METHOD AND MEANS FOR SELECTING PEPTIDES AND PROTEINS WITH SPECIFIC AFFINITY TO A TARGET MOLECULE |
-
1991
- 1991-12-03 AT AT92902109T patent/ATE164395T1/en not_active IP Right Cessation
- 1991-12-03 DE DE69129154T patent/DE69129154T2/en not_active Expired - Lifetime
- 1991-12-03 DK DK92902109T patent/DK0564531T3/en active
- 1991-12-03 EP EP92902109A patent/EP0564531B1/en not_active Expired - Lifetime
- 1991-12-03 CA CA002405246A patent/CA2405246A1/en not_active Abandoned
- 1991-12-03 ES ES92902109T patent/ES2113940T3/en not_active Expired - Lifetime
- 1991-12-03 CA CA002095633A patent/CA2095633C/en not_active Expired - Lifetime
- 1991-12-03 WO PCT/US1991/009133 patent/WO1992009690A2/en active IP Right Grant
- 1991-12-03 US US08/050,058 patent/US5750373A/en not_active Expired - Lifetime
-
1995
- 1995-06-05 US US08/463,587 patent/US5821047A/en not_active Expired - Lifetime
- 1995-06-05 US US08/463,667 patent/US5834598A/en not_active Expired - Lifetime
-
1997
- 1997-09-03 US US08/923,854 patent/US6040136A/en not_active Expired - Fee Related
-
1998
- 1998-03-27 GR GR980400652T patent/GR3026468T3/en unknown
-
2005
- 2005-08-08 US US11/199,062 patent/US20060115874A1/en not_active Abandoned
-
2007
- 2007-06-11 US US11/761,180 patent/US20080038717A1/en not_active Abandoned
-
2009
- 2009-07-24 US US12/508,859 patent/US20100035236A1/en not_active Abandoned
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080194477A1 (en) * | 2002-09-09 | 2008-08-14 | Rene Gantier | Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules |
US7884073B2 (en) | 2004-11-04 | 2011-02-08 | Hanall Biopharma Co., Ltd. | Modified growth hormone |
US7998930B2 (en) | 2004-11-04 | 2011-08-16 | Hanall Biopharma Co., Ltd. | Modified growth hormones |
US8222209B2 (en) | 2004-11-04 | 2012-07-17 | Hanall Biopharma Co., Ltd. | Modified growth hormones that exhibit increased protease resistance and pharmaceutical compositions thereof |
US8679490B2 (en) | 2005-11-07 | 2014-03-25 | Genentech, Inc. | Binding polypeptides with diversified and consensus VH/VL hypervariable sequences |
US20070237764A1 (en) * | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US8937159B2 (en) | 2009-12-16 | 2015-01-20 | Abbvie Biotherapeutics Inc. | Anti-HER2 antibodies and their uses |
US20110177095A1 (en) * | 2009-12-16 | 2011-07-21 | Abbott Biotherapeutics Corporation | Anti-her2 antibodies and their uses |
US9078878B2 (en) | 2010-12-01 | 2015-07-14 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9783602B2 (en) | 2010-12-01 | 2017-10-10 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
US8911734B2 (en) | 2010-12-01 | 2014-12-16 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9539324B2 (en) | 2010-12-01 | 2017-01-10 | Alderbio Holdings, Llc | Methods of preventing inflammation and treating pain using anti-NGF compositions |
US9718882B2 (en) | 2010-12-01 | 2017-08-01 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with P75 |
US9738713B2 (en) | 2010-12-01 | 2017-08-22 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies |
US9783601B2 (en) | 2010-12-01 | 2017-10-10 | Alderbio Holdings Llc | Methods of preventing inflammation and treating pain using anti-NGF compositions |
US9067988B2 (en) | 2010-12-01 | 2015-06-30 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies |
US9884909B2 (en) | 2010-12-01 | 2018-02-06 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
US10221236B2 (en) | 2010-12-01 | 2019-03-05 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TRKA without affecting the association of NGF with P75 |
US10227402B2 (en) | 2010-12-01 | 2019-03-12 | Alderbio Holdings Llc | Anti-NGF antibodies and anti-NGF antibody fragments |
US10344083B2 (en) | 2010-12-01 | 2019-07-09 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
US10457727B2 (en) | 2010-12-01 | 2019-10-29 | Alderbio Holdings Llc | Methods of preventing inflammation and treating pain using anti-NGF compositions |
US11214610B2 (en) | 2010-12-01 | 2022-01-04 | H. Lundbeck A/S | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris |
Also Published As
Publication number | Publication date |
---|---|
US6040136A (en) | 2000-03-21 |
CA2095633A1 (en) | 1992-06-04 |
US5834598A (en) | 1998-11-10 |
EP0564531B1 (en) | 1998-03-25 |
CA2405246A1 (en) | 1992-06-11 |
ES2113940T3 (en) | 1998-05-16 |
DE69129154T2 (en) | 1998-08-20 |
WO1992009690A2 (en) | 1992-06-11 |
CA2095633C (en) | 2003-02-04 |
WO1992009690A3 (en) | 1992-12-10 |
ATE164395T1 (en) | 1998-04-15 |
GR3026468T3 (en) | 1998-06-30 |
US20060115874A1 (en) | 2006-06-01 |
US5821047A (en) | 1998-10-13 |
EP0564531A1 (en) | 1993-10-13 |
US5750373A (en) | 1998-05-12 |
DK0564531T3 (en) | 1998-09-28 |
DE69129154D1 (en) | 1998-04-30 |
US20100035236A1 (en) | 2010-02-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5834598A (en) | Human growth hormone variants | |
US5688666A (en) | Growth hormone variants with altered binding properties | |
US5780279A (en) | Method of selection of proteolytic cleavage sites by directed evolution and phagemid display | |
CA2001774C (en) | Method for identifying active domains and amino acid residues in polypeptides and hormone variants | |
Lowman et al. | Selecting high-affinity binding proteins by monovalent phage display | |
US5534617A (en) | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 | |
US6013478A (en) | Method for identifying active domains and amino acid residues in polypeptides and hormone variants | |
US6057098A (en) | Polyvalent display libraries | |
JP2006104205A (en) | Human growth hormone variant | |
Gram et al. | Phage display as a rapid gene expression system: production of bioactive cytokine-phage and generation of neutralizing monoclonal antibodies | |
WO1998047343A9 (en) | Antibodies or binding protein libraries displayed on phage, cells, or other replicatable genetic packages | |
WO1995015982A2 (en) | Process for generating specific antibodies | |
WO1995011922A1 (en) | In vitro peptide and antibody display libraries | |
JP2001503131A (en) | Compositions and methods for screening pharmaceutical libraries | |
JP3765959B2 (en) | Hormone variant |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |