US20100189682A1 - Biologically active proteins having increased In Vivo and/or In Vitro stability - Google Patents
Biologically active proteins having increased In Vivo and/or In Vitro stability Download PDFInfo
- Publication number
- US20100189682A1 US20100189682A1 US12/634,556 US63455609A US2010189682A1 US 20100189682 A1 US20100189682 A1 US 20100189682A1 US 63455609 A US63455609 A US 63455609A US 2010189682 A1 US2010189682 A1 US 2010189682A1
- Authority
- US
- United States
- Prior art keywords
- protein
- sequences
- urp
- alpha
- channel
- 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 332
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 304
- 230000001965 increasing effect Effects 0.000 title claims description 20
- 238000000338 in vitro Methods 0.000 title claims description 6
- 238000001727 in vivo Methods 0.000 title claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 101
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 62
- 239000000203 mixture Substances 0.000 claims abstract description 32
- 229920001184 polypeptide Polymers 0.000 claims abstract description 28
- 239000013598 vector Substances 0.000 claims abstract description 18
- 235000018102 proteins Nutrition 0.000 claims description 288
- 235000001014 amino acid Nutrition 0.000 claims description 142
- 150000001413 amino acids Chemical class 0.000 claims description 136
- 230000027455 binding Effects 0.000 claims description 117
- 238000000034 method Methods 0.000 claims description 82
- 210000004027 cell Anatomy 0.000 claims description 43
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 33
- 206010028980 Neoplasm Diseases 0.000 claims description 30
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 19
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 18
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 9
- 235000004279 alanine Nutrition 0.000 claims description 9
- 125000000539 amino acid group Chemical group 0.000 claims description 9
- 230000004071 biological effect Effects 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 229940088597 hormone Drugs 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 7
- 239000005556 hormone Substances 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 6
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 5
- 239000003102 growth factor Substances 0.000 claims description 5
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 4
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 4
- 239000000854 Human Growth Hormone Substances 0.000 claims description 4
- 108010047761 Interferon-alpha Proteins 0.000 claims description 4
- 102000006992 Interferon-alpha Human genes 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 208000023275 Autoimmune disease Diseases 0.000 claims description 3
- 208000035473 Communicable disease Diseases 0.000 claims description 3
- 102000003951 Erythropoietin Human genes 0.000 claims description 3
- 108090000394 Erythropoietin Proteins 0.000 claims description 3
- 229940105423 erythropoietin Drugs 0.000 claims description 3
- 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 3
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 claims description 3
- 108010054218 Factor VIII Proteins 0.000 claims description 2
- 102000001690 Factor VIII Human genes 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 230000007812 deficiency Effects 0.000 claims description 2
- 239000000122 growth hormone Substances 0.000 claims description 2
- 102000003390 tumor necrosis factor Human genes 0.000 claims 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims 1
- 102000003996 Interferon-beta Human genes 0.000 claims 1
- 108090000467 Interferon-beta Proteins 0.000 claims 1
- 102000008070 Interferon-gamma Human genes 0.000 claims 1
- 108010074328 Interferon-gamma Proteins 0.000 claims 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 claims 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 claims 1
- 102000019298 Lipocalin Human genes 0.000 claims 1
- 108050006654 Lipocalin Proteins 0.000 claims 1
- 102000013519 Lipocalin-2 Human genes 0.000 claims 1
- 108010051335 Lipocalin-2 Proteins 0.000 claims 1
- 206010063837 Reperfusion injury Diseases 0.000 claims 1
- 208000007536 Thrombosis Diseases 0.000 claims 1
- 208000007502 anemia Diseases 0.000 claims 1
- 229940105778 coagulation factor viii Drugs 0.000 claims 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims 1
- 206010012601 diabetes mellitus Diseases 0.000 claims 1
- 208000035475 disorder Diseases 0.000 claims 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims 1
- 229940044627 gamma-interferon Drugs 0.000 claims 1
- 230000002458 infectious effect Effects 0.000 claims 1
- 208000027866 inflammatory disease Diseases 0.000 claims 1
- 208000017169 kidney disease Diseases 0.000 claims 1
- 208000010125 myocardial infarction Diseases 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 230000002035 prolonged effect Effects 0.000 claims 1
- 229920000642 polymer Polymers 0.000 abstract description 34
- 230000002068 genetic effect Effects 0.000 abstract description 19
- 231100000765 toxin Toxicity 0.000 abstract description 16
- 108700012359 toxins Proteins 0.000 abstract description 16
- 239000003053 toxin Substances 0.000 abstract description 15
- 229940024606 amino acid Drugs 0.000 description 141
- 241000282414 Homo sapiens Species 0.000 description 119
- -1 ErbB2 Proteins 0.000 description 98
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 94
- 239000000047 product Substances 0.000 description 68
- 102000035195 Peptidases Human genes 0.000 description 59
- 108091005804 Peptidases Proteins 0.000 description 59
- 239000004365 Protease Substances 0.000 description 59
- 238000013459 approach Methods 0.000 description 52
- 239000003795 chemical substances by application Substances 0.000 description 49
- 239000004471 Glycine Substances 0.000 description 45
- 210000001519 tissue Anatomy 0.000 description 40
- 235000018417 cysteine Nutrition 0.000 description 39
- 239000012636 effector Substances 0.000 description 37
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 36
- 235000019419 proteases Nutrition 0.000 description 35
- 239000003814 drug Substances 0.000 description 32
- 229940079593 drug Drugs 0.000 description 32
- 238000013461 design Methods 0.000 description 31
- 230000005847 immunogenicity Effects 0.000 description 29
- 210000002966 serum Anatomy 0.000 description 29
- 108020004705 Codon Proteins 0.000 description 27
- 108020004414 DNA Proteins 0.000 description 27
- 238000004519 manufacturing process Methods 0.000 description 27
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 25
- 102000003839 Human Proteins Human genes 0.000 description 24
- 108090000144 Human Proteins Proteins 0.000 description 24
- 102000040430 polynucleotide Human genes 0.000 description 24
- 108091033319 polynucleotide Proteins 0.000 description 24
- 239000002157 polynucleotide Substances 0.000 description 24
- 210000001744 T-lymphocyte Anatomy 0.000 description 21
- 230000003993 interaction Effects 0.000 description 21
- 239000012528 membrane Substances 0.000 description 21
- 230000021615 conjugation Effects 0.000 description 19
- 239000012634 fragment Substances 0.000 description 19
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 18
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 18
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 18
- 238000002347 injection Methods 0.000 description 18
- 239000007924 injection Substances 0.000 description 18
- 235000004400 serine Nutrition 0.000 description 18
- 239000000126 substance Substances 0.000 description 18
- 102000001189 Cyclic Peptides Human genes 0.000 description 17
- 108010069514 Cyclic Peptides Proteins 0.000 description 17
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 17
- 210000004899 c-terminal region Anatomy 0.000 description 17
- 230000004927 fusion Effects 0.000 description 17
- 239000004473 Threonine Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 230000002829 reductive effect Effects 0.000 description 16
- 102000004506 Blood Proteins Human genes 0.000 description 15
- 108010017384 Blood Proteins Proteins 0.000 description 15
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 15
- 230000006870 function Effects 0.000 description 15
- 230000001976 improved effect Effects 0.000 description 15
- 239000003446 ligand Substances 0.000 description 15
- 238000004091 panning Methods 0.000 description 15
- 102000005962 receptors Human genes 0.000 description 15
- 108020003175 receptors Proteins 0.000 description 15
- 235000008521 threonine Nutrition 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 14
- 229930195712 glutamate Natural products 0.000 description 14
- 125000003729 nucleotide group Chemical group 0.000 description 14
- 238000002823 phage display Methods 0.000 description 14
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 13
- 235000013930 proline Nutrition 0.000 description 13
- 238000000746 purification Methods 0.000 description 13
- 230000003252 repetitive effect Effects 0.000 description 13
- 238000012216 screening Methods 0.000 description 13
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 12
- 108091034117 Oligonucleotide Proteins 0.000 description 12
- 108010029485 Protein Isoforms Proteins 0.000 description 12
- 102000001708 Protein Isoforms Human genes 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 238000004422 calculation algorithm Methods 0.000 description 12
- 230000002209 hydrophobic effect Effects 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 150000002019 disulfides Chemical class 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 230000008569 process Effects 0.000 description 11
- 108091006146 Channels Proteins 0.000 description 10
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 10
- 101000610604 Homo sapiens Tumor necrosis factor receptor superfamily member 10B Proteins 0.000 description 10
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 10
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 10
- 102100040112 Tumor necrosis factor receptor superfamily member 10B Human genes 0.000 description 10
- 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 10
- 229940009098 aspartate Drugs 0.000 description 10
- 239000011324 bead Substances 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 230000006320 pegylation Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 102100024598 Tumor necrosis factor ligand superfamily member 10 Human genes 0.000 description 9
- 102100036922 Tumor necrosis factor ligand superfamily member 13B Human genes 0.000 description 9
- 102100040113 Tumor necrosis factor receptor superfamily member 10A Human genes 0.000 description 9
- 239000000427 antigen Substances 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 239000000539 dimer Substances 0.000 description 9
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 230000004962 physiological condition Effects 0.000 description 9
- 238000012545 processing Methods 0.000 description 9
- 239000004475 Arginine Substances 0.000 description 8
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 8
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 8
- 101100369992 Homo sapiens TNFSF10 gene Proteins 0.000 description 8
- 101000610605 Homo sapiens Tumor necrosis factor receptor superfamily member 10A Proteins 0.000 description 8
- 108090000862 Ion Channels Proteins 0.000 description 8
- 102000004310 Ion Channels Human genes 0.000 description 8
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 8
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 8
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 8
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 8
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 108700012411 TNFSF10 Proteins 0.000 description 8
- 102100033142 Transcription factor 20 Human genes 0.000 description 8
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 8
- 238000007792 addition Methods 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 238000004132 cross linking Methods 0.000 description 8
- 102000037865 fusion proteins Human genes 0.000 description 8
- 108020001507 fusion proteins Proteins 0.000 description 8
- 239000005090 green fluorescent protein Substances 0.000 description 8
- 230000028993 immune response Effects 0.000 description 8
- 229960000310 isoleucine Drugs 0.000 description 8
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 8
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 8
- 230000017854 proteolysis Effects 0.000 description 8
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 8
- 239000004474 valine Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 102100032937 CD40 ligand Human genes 0.000 description 7
- 102000000844 Cell Surface Receptors Human genes 0.000 description 7
- 108010001857 Cell Surface Receptors Proteins 0.000 description 7
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 108700026244 Open Reading Frames Proteins 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 7
- 235000009697 arginine Nutrition 0.000 description 7
- 229960003272 asparaginase Drugs 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 239000003431 cross linking reagent Substances 0.000 description 7
- 238000006471 dimerization reaction Methods 0.000 description 7
- 230000006334 disulfide bridging Effects 0.000 description 7
- 235000018977 lysine Nutrition 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 230000002685 pulmonary effect Effects 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 230000002588 toxic effect Effects 0.000 description 7
- 229940124024 weight reducing agent Drugs 0.000 description 7
- 102100034571 AT-rich interactive domain-containing protein 1B Human genes 0.000 description 6
- 108010059616 Activins Proteins 0.000 description 6
- 102000005606 Activins Human genes 0.000 description 6
- 108010024976 Asparaginase Proteins 0.000 description 6
- 102000015790 Asparaginase Human genes 0.000 description 6
- 108700010070 Codon Usage Proteins 0.000 description 6
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 6
- 108090000852 Forkhead Transcription Factors Proteins 0.000 description 6
- 102000004315 Forkhead Transcription Factors Human genes 0.000 description 6
- 101000924255 Homo sapiens AT-rich interactive domain-containing protein 1B Proteins 0.000 description 6
- 101000666661 Homo sapiens Rho-related GTP-binding protein RhoU Proteins 0.000 description 6
- 101000801228 Homo sapiens Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 6
- 102100020873 Interleukin-2 Human genes 0.000 description 6
- 108010002350 Interleukin-2 Proteins 0.000 description 6
- 239000004472 Lysine Substances 0.000 description 6
- 108010052285 Membrane Proteins Proteins 0.000 description 6
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 6
- 102100038399 Rho-related GTP-binding protein RhoU Human genes 0.000 description 6
- 102100035600 Synergin gamma Human genes 0.000 description 6
- 101710139420 THO complex subunit 4 Proteins 0.000 description 6
- 102100036434 THO complex subunit 4 Human genes 0.000 description 6
- 101710119730 Transcription factor 20 Proteins 0.000 description 6
- 101710181056 Tumor necrosis factor ligand superfamily member 13B Proteins 0.000 description 6
- 102100033732 Tumor necrosis factor receptor superfamily member 1A Human genes 0.000 description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 6
- 239000000488 activin Substances 0.000 description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 6
- 235000003704 aspartic acid Nutrition 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 239000003638 chemical reducing agent Substances 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 235000013922 glutamic acid Nutrition 0.000 description 6
- 239000004220 glutamic acid Substances 0.000 description 6
- 230000008105 immune reaction Effects 0.000 description 6
- 238000010348 incorporation Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 230000009467 reduction Effects 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 231100000331 toxic Toxicity 0.000 description 6
- 102100027207 CD27 antigen Human genes 0.000 description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 239000004971 Cross linker Substances 0.000 description 5
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 5
- 241001524679 Escherichia virus M13 Species 0.000 description 5
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 5
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 5
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 5
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 5
- 101000679903 Homo sapiens Tumor necrosis factor receptor superfamily member 25 Proteins 0.000 description 5
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 5
- 102000007999 Nuclear Proteins Human genes 0.000 description 5
- 108010089610 Nuclear Proteins Proteins 0.000 description 5
- 102100039019 Nuclear receptor subfamily 0 group B member 1 Human genes 0.000 description 5
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 5
- 102100040247 Tumor necrosis factor Human genes 0.000 description 5
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 5
- 102100022203 Tumor necrosis factor receptor superfamily member 25 Human genes 0.000 description 5
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 5
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 5
- 230000002776 aggregation Effects 0.000 description 5
- 238000004220 aggregation Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 239000002340 cardiotoxin Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 108091005573 modified proteins Proteins 0.000 description 5
- 102000035118 modified proteins Human genes 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 231100000219 mutagenic Toxicity 0.000 description 5
- 230000003505 mutagenic effect Effects 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- 230000006798 recombination Effects 0.000 description 5
- 238000005215 recombination Methods 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- 102100038074 5'-AMP-activated protein kinase subunit beta-1 Human genes 0.000 description 4
- 108010029697 CD40 Ligand Proteins 0.000 description 4
- 108010032088 Calpain Proteins 0.000 description 4
- 102000007590 Calpain Human genes 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 102000009058 Death Domain Receptors Human genes 0.000 description 4
- 108010049207 Death Domain Receptors Proteins 0.000 description 4
- 102100029727 Enteropeptidase Human genes 0.000 description 4
- 108010013369 Enteropeptidase Proteins 0.000 description 4
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 4
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 4
- 101000603323 Homo sapiens Nuclear receptor subfamily 0 group B member 1 Proteins 0.000 description 4
- 101001077488 Homo sapiens RNA-binding protein Raly Proteins 0.000 description 4
- 101000659071 Homo sapiens Synergin gamma Proteins 0.000 description 4
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 4
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 4
- 101000679921 Homo sapiens Tumor necrosis factor receptor superfamily member 21 Proteins 0.000 description 4
- 101000597785 Homo sapiens Tumor necrosis factor receptor superfamily member 6B Proteins 0.000 description 4
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 4
- 102100022905 Keratin, type II cytoskeletal 1 Human genes 0.000 description 4
- 102100022819 MHC class II regulatory factor RFX1 Human genes 0.000 description 4
- 102000018697 Membrane Proteins Human genes 0.000 description 4
- 101000783356 Naja sputatrix Cytotoxin Proteins 0.000 description 4
- 102100025052 RNA-binding protein Raly Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 102100038707 Small nuclear ribonucleoprotein Sm D1 Human genes 0.000 description 4
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 108020004566 Transfer RNA Proteins 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 102100032100 Tumor necrosis factor ligand superfamily member 8 Human genes 0.000 description 4
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 4
- 102100033726 Tumor necrosis factor receptor superfamily member 17 Human genes 0.000 description 4
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 4
- 102100022205 Tumor necrosis factor receptor superfamily member 21 Human genes 0.000 description 4
- 102100035284 Tumor necrosis factor receptor superfamily member 6B Human genes 0.000 description 4
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 4
- 231100000677 cardiotoxin Toxicity 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229920001477 hydrophilic polymer Polymers 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 108020001580 protein domains Proteins 0.000 description 4
- 230000002797 proteolythic effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 210000003705 ribosome Anatomy 0.000 description 4
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- VILFTWLXLYIEMV-UHFFFAOYSA-N 1,5-difluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC([N+]([O-])=O)=C(F)C=C1F VILFTWLXLYIEMV-UHFFFAOYSA-N 0.000 description 3
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical class C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 3
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 3
- 102100032187 Androgen receptor Human genes 0.000 description 3
- 102000004452 Arginase Human genes 0.000 description 3
- 108700024123 Arginases Proteins 0.000 description 3
- 102100030009 Azurocidin Human genes 0.000 description 3
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 3
- 102100025277 C-X-C motif chemokine 13 Human genes 0.000 description 3
- 101150013553 CD40 gene Proteins 0.000 description 3
- 102100032912 CD44 antigen Human genes 0.000 description 3
- 102000000905 Cadherin Human genes 0.000 description 3
- 108050007957 Cadherin Proteins 0.000 description 3
- 108060002063 Cyclotide Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 102100029792 Dentin sialophosphoprotein Human genes 0.000 description 3
- 102100020743 Dipeptidase 1 Human genes 0.000 description 3
- 108090000204 Dipeptidase 1 Proteins 0.000 description 3
- 102100020751 Dipeptidyl peptidase 2 Human genes 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102100037354 Ectodysplasin-A Human genes 0.000 description 3
- 101150064015 FAS gene Proteins 0.000 description 3
- 102100028466 Frizzled-8 Human genes 0.000 description 3
- 102000054184 GADD45 Human genes 0.000 description 3
- 102000010956 Glypican Human genes 0.000 description 3
- 108050001154 Glypican Proteins 0.000 description 3
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 description 3
- 101000858064 Homo sapiens C-X-C motif chemokine 13 Proteins 0.000 description 3
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 description 3
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 3
- 101000880080 Homo sapiens Ectodysplasin-A Proteins 0.000 description 3
- 101001066158 Homo sapiens Growth arrest and DNA damage-inducible protein GADD45 alpha Proteins 0.000 description 3
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 description 3
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 101000702545 Homo sapiens Transcription activator BRG1 Proteins 0.000 description 3
- 101000830600 Homo sapiens Tumor necrosis factor ligand superfamily member 13 Proteins 0.000 description 3
- 101000597779 Homo sapiens Tumor necrosis factor ligand superfamily member 18 Proteins 0.000 description 3
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 3
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 3
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 102100025390 Integrin beta-2 Human genes 0.000 description 3
- 108010065805 Interleukin-12 Proteins 0.000 description 3
- 102000013462 Interleukin-12 Human genes 0.000 description 3
- 108010065637 Interleukin-23 Proteins 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 102000015696 Interleukins Human genes 0.000 description 3
- 108010063738 Interleukins Proteins 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 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
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 3
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 3
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 3
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 3
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 description 3
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 3
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 3
- 102100023832 Prolyl endopeptidase FAP Human genes 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 102000018968 Salivary Cystatins Human genes 0.000 description 3
- 108010026774 Salivary Cystatins Proteins 0.000 description 3
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 108020005038 Terminator Codon Proteins 0.000 description 3
- 102100031027 Transcription activator BRG1 Human genes 0.000 description 3
- 102100035715 Transcriptional activator protein Pur-alpha Human genes 0.000 description 3
- 102100024584 Tumor necrosis factor ligand superfamily member 12 Human genes 0.000 description 3
- 102100024585 Tumor necrosis factor ligand superfamily member 13 Human genes 0.000 description 3
- 102100035283 Tumor necrosis factor ligand superfamily member 18 Human genes 0.000 description 3
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 3
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 3
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000030741 antigen processing and presentation Effects 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000030833 cell death Effects 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 108010088492 dentin sialophosphoprotein Proteins 0.000 description 3
- 229960004679 doxorubicin Drugs 0.000 description 3
- 230000002500 effect on skin Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 102000012803 ephrin Human genes 0.000 description 3
- 108060002566 ephrin Proteins 0.000 description 3
- 210000000744 eyelid Anatomy 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 210000003128 head Anatomy 0.000 description 3
- 125000001165 hydrophobic group Chemical group 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 229960000485 methotrexate Drugs 0.000 description 3
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000003471 mutagenic agent Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 230000009871 nonspecific binding Effects 0.000 description 3
- 230000035515 penetration Effects 0.000 description 3
- 108050009312 plexin Proteins 0.000 description 3
- 102000002022 plexin Human genes 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 229940002612 prodrug Drugs 0.000 description 3
- 239000000651 prodrug Substances 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 231100001274 therapeutic index Toxicity 0.000 description 3
- 230000001960 triggered effect Effects 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- BVGLZNQZEYAYBJ-QWZQWHGGSA-N α-cobratoxin Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CS)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)CC1=CC=C(O)C=C1 BVGLZNQZEYAYBJ-QWZQWHGGSA-N 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- IEUUDEWWMRQUDS-UHFFFAOYSA-N (6-azaniumylidene-1,6-dimethoxyhexylidene)azanium;dichloride Chemical compound Cl.Cl.COC(=N)CCCCC(=N)OC IEUUDEWWMRQUDS-UHFFFAOYSA-N 0.000 description 2
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 2
- FMYBFLOWKQRBST-UHFFFAOYSA-N 2-[bis(carboxymethyl)amino]acetic acid;nickel Chemical compound [Ni].OC(=O)CN(CC(O)=O)CC(O)=O FMYBFLOWKQRBST-UHFFFAOYSA-N 0.000 description 2
- 108010082808 4-1BB Ligand Proteins 0.000 description 2
- 102100029769 ADAMTS-like protein 1 Human genes 0.000 description 2
- 101710159080 Aconitate hydratase A Proteins 0.000 description 2
- 101710159078 Aconitate hydratase B Proteins 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 108010075348 Activated-Leukocyte Cell Adhesion Molecule Proteins 0.000 description 2
- 102100031786 Adiponectin Human genes 0.000 description 2
- 108010076365 Adiponectin Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102100022749 Aminopeptidase N Human genes 0.000 description 2
- 102000007299 Amphiregulin Human genes 0.000 description 2
- 108010033760 Amphiregulin Proteins 0.000 description 2
- 102100022987 Angiogenin Human genes 0.000 description 2
- 102100025665 Angiopoietin-related protein 1 Human genes 0.000 description 2
- 102100034598 Angiopoietin-related protein 7 Human genes 0.000 description 2
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 2
- 102100027139 Ankyrin repeat and SAM domain-containing protein 1A Human genes 0.000 description 2
- 102000004145 Annexin A1 Human genes 0.000 description 2
- 108090000663 Annexin A1 Proteins 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 2
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 2
- 102100038238 Aromatic-L-amino-acid decarboxylase Human genes 0.000 description 2
- CJLHTKGWEUGORV-UHFFFAOYSA-N Artemin Chemical compound C1CC2(C)C(O)CCC(=C)C2(O)C2C1C(C)C(=O)O2 CJLHTKGWEUGORV-UHFFFAOYSA-N 0.000 description 2
- 102100022146 Arylsulfatase A Human genes 0.000 description 2
- UKGGPJNBONZZCM-WDSKDSINSA-N Asp-Pro Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O UKGGPJNBONZZCM-WDSKDSINSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100022718 Atypical chemokine receptor 2 Human genes 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 101710154607 Azurocidin Proteins 0.000 description 2
- 108010028006 B-Cell Activating Factor Proteins 0.000 description 2
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 2
- 102100037150 BMP and activin membrane-bound inhibitor homolog Human genes 0.000 description 2
- 102100024507 BMP-2-inducible protein kinase Human genes 0.000 description 2
- 102100028239 Basal cell adhesion molecule Human genes 0.000 description 2
- 102100036597 Basement membrane-specific heparan sulfate proteoglycan core protein Human genes 0.000 description 2
- 102100032305 Bcl-2 homologous antagonist/killer Human genes 0.000 description 2
- KXDAEFPNCMNJSK-UHFFFAOYSA-N Benzamide Chemical compound NC(=O)C1=CC=CC=C1 KXDAEFPNCMNJSK-UHFFFAOYSA-N 0.000 description 2
- 102100038495 Bile acid receptor Human genes 0.000 description 2
- 102100028728 Bone morphogenetic protein 1 Human genes 0.000 description 2
- 108090000654 Bone morphogenetic protein 1 Proteins 0.000 description 2
- 102000003928 Bone morphogenetic protein 15 Human genes 0.000 description 2
- 108090000349 Bone morphogenetic protein 15 Proteins 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 102100036849 C-C motif chemokine 24 Human genes 0.000 description 2
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 2
- 102100021936 C-C motif chemokine 27 Human genes 0.000 description 2
- 102100025250 C-X-C motif chemokine 14 Human genes 0.000 description 2
- 102100039396 C-X-C motif chemokine 16 Human genes 0.000 description 2
- 102100039398 C-X-C motif chemokine 2 Human genes 0.000 description 2
- 102100036153 C-X-C motif chemokine 6 Human genes 0.000 description 2
- 102100032752 C-reactive protein Human genes 0.000 description 2
- 102100028667 C-type lectin domain family 4 member A Human genes 0.000 description 2
- 102100028665 C-type lectin domain family 4 member F Human genes 0.000 description 2
- 102100040843 C-type lectin domain family 4 member M Human genes 0.000 description 2
- 102100040839 C-type lectin domain family 6 member A Human genes 0.000 description 2
- 102100040840 C-type lectin domain family 7 member A Human genes 0.000 description 2
- 101150049756 CCL6 gene Proteins 0.000 description 2
- 102100031168 CCN family member 2 Human genes 0.000 description 2
- 108010049990 CD13 Antigens Proteins 0.000 description 2
- 102100024210 CD166 antigen Human genes 0.000 description 2
- 102100038078 CD276 antigen Human genes 0.000 description 2
- 101710185679 CD276 antigen Proteins 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 2
- 108010017987 CD30 Ligand Proteins 0.000 description 2
- 102100036008 CD48 antigen Human genes 0.000 description 2
- 108010084313 CD58 Antigens Proteins 0.000 description 2
- 102100022002 CD59 glycoprotein Human genes 0.000 description 2
- 102100025221 CD70 antigen Human genes 0.000 description 2
- 102100028228 COUP transcription factor 1 Human genes 0.000 description 2
- 102100028226 COUP transcription factor 2 Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 102100029758 Cadherin-4 Human genes 0.000 description 2
- 102000004631 Calcineurin Human genes 0.000 description 2
- 108010042955 Calcineurin Proteins 0.000 description 2
- 101000724917 Calliophis bivirgatus Delta-elapitoxin-Cb1a Proteins 0.000 description 2
- 101000724912 Calliophis bivirgatus Maticotoxin A Proteins 0.000 description 2
- 102100036214 Cannabinoid receptor 2 Human genes 0.000 description 2
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 2
- 108010080937 Carboxypeptidases A Proteins 0.000 description 2
- 102000000496 Carboxypeptidases A Human genes 0.000 description 2
- 102100027473 Cartilage oligomeric matrix protein Human genes 0.000 description 2
- 108010076667 Caspases Proteins 0.000 description 2
- 108010059081 Cathepsin A Proteins 0.000 description 2
- 102000005572 Cathepsin A Human genes 0.000 description 2
- 102100024940 Cathepsin K Human genes 0.000 description 2
- 102100027835 Cell death regulator Aven Human genes 0.000 description 2
- 102100037328 Chitotriosidase-1 Human genes 0.000 description 2
- 102100023460 Choline O-acetyltransferase Human genes 0.000 description 2
- 108010058699 Choline O-acetyltransferase Proteins 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 108010086232 Cobra Neurotoxin Proteins Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 102100024330 Collectin-12 Human genes 0.000 description 2
- 102100024342 Contactin-2 Human genes 0.000 description 2
- 102100022786 Creatine kinase M-type Human genes 0.000 description 2
- 102100029141 Cyclic nucleotide-gated cation channel beta-1 Human genes 0.000 description 2
- 102000015833 Cystatin Human genes 0.000 description 2
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 2
- 108010075031 Cytochromes c Proteins 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 2
- 101000724921 Dendroaspis polylepis polylepis Dendroaspis polylepis MT9 Proteins 0.000 description 2
- 108010082610 Deoxyribonuclease (Pyrimidine Dimer) Proteins 0.000 description 2
- 102100034579 Desmoglein-1 Human genes 0.000 description 2
- 101000876610 Dictyostelium discoideum Extracellular signal-regulated kinase 2 Proteins 0.000 description 2
- 102000017944 Dishevelled Human genes 0.000 description 2
- 108050007016 Dishevelled Proteins 0.000 description 2
- 102100031111 Disintegrin and metalloproteinase domain-containing protein 17 Human genes 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 102100021598 Endoplasmic reticulum aminopeptidase 1 Human genes 0.000 description 2
- 102100021597 Endoplasmic reticulum aminopeptidase 2 Human genes 0.000 description 2
- 102100030751 Eomesodermin homolog Human genes 0.000 description 2
- 102100023688 Eotaxin Human genes 0.000 description 2
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 2
- 102100029951 Estrogen receptor beta Human genes 0.000 description 2
- 102100035977 Exostosin-like 2 Human genes 0.000 description 2
- 102100035976 Exostosin-like 3 Human genes 0.000 description 2
- 101150021185 FGF gene Proteins 0.000 description 2
- 108010014173 Factor X Proteins 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 102100031517 Fc receptor-like protein 1 Human genes 0.000 description 2
- 102100031511 Fc receptor-like protein 2 Human genes 0.000 description 2
- 102100031513 Fc receptor-like protein 4 Human genes 0.000 description 2
- 102100031507 Fc receptor-like protein 5 Human genes 0.000 description 2
- 102100035308 Fibroblast growth factor 17 Human genes 0.000 description 2
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 2
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 201000008808 Fibrosarcoma Diseases 0.000 description 2
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 2
- 102100020997 Fractalkine Human genes 0.000 description 2
- 101710140933 Frizzled-8 Proteins 0.000 description 2
- 108010001517 Galectin 3 Proteins 0.000 description 2
- 102100039558 Galectin-3 Human genes 0.000 description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- 102000051325 Glucagon Human genes 0.000 description 2
- 108060003199 Glucagon Proteins 0.000 description 2
- 102100035902 Glutamate decarboxylase 1 Human genes 0.000 description 2
- 102100035857 Glutamate decarboxylase 2 Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 108010041881 Growth Differentiation Factor 10 Proteins 0.000 description 2
- 102100034221 Growth-regulated alpha protein Human genes 0.000 description 2
- 102100040895 Growth/differentiation factor 10 Human genes 0.000 description 2
- 101710081758 High affinity cationic amino acid transporter 1 Proteins 0.000 description 2
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 102100035043 Histone-lysine N-methyltransferase EHMT1 Human genes 0.000 description 2
- 108700005087 Homeobox Genes Proteins 0.000 description 2
- 102100030968 Homeobox even-skipped homolog protein 2 Human genes 0.000 description 2
- 101710194869 Homeobox even-skipped homolog protein 2 Proteins 0.000 description 2
- 102100028411 Homeobox protein Hox-B3 Human genes 0.000 description 2
- 102100039545 Homeobox protein Hox-D11 Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000924546 Homo sapiens Angiopoietin-related protein 7 Proteins 0.000 description 2
- 101000694621 Homo sapiens Ankyrin repeat and SAM domain-containing protein 1A Proteins 0.000 description 2
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 2
- 101000678892 Homo sapiens Atypical chemokine receptor 2 Proteins 0.000 description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 2
- 101100325746 Homo sapiens BAK1 gene Proteins 0.000 description 2
- 101000740070 Homo sapiens BMP and activin membrane-bound inhibitor homolog Proteins 0.000 description 2
- 101000762370 Homo sapiens BMP-2-inducible protein kinase Proteins 0.000 description 2
- 101000935638 Homo sapiens Basal cell adhesion molecule Proteins 0.000 description 2
- 101000603876 Homo sapiens Bile acid receptor Proteins 0.000 description 2
- 101000766294 Homo sapiens Branched-chain-amino-acid aminotransferase, mitochondrial Proteins 0.000 description 2
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 2
- 101000858068 Homo sapiens C-X-C motif chemokine 14 Proteins 0.000 description 2
- 101000889133 Homo sapiens C-X-C motif chemokine 16 Proteins 0.000 description 2
- 101000766908 Homo sapiens C-type lectin domain family 4 member A Proteins 0.000 description 2
- 101000766920 Homo sapiens C-type lectin domain family 4 member F Proteins 0.000 description 2
- 101000749311 Homo sapiens C-type lectin domain family 4 member M Proteins 0.000 description 2
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 description 2
- 101000771075 Homo sapiens Cyclic nucleotide-gated cation channel beta-1 Proteins 0.000 description 2
- 101001010910 Homo sapiens Estrogen receptor beta Proteins 0.000 description 2
- 101000846911 Homo sapiens Fc receptor-like protein 2 Proteins 0.000 description 2
- 101000846909 Homo sapiens Fc receptor-like protein 4 Proteins 0.000 description 2
- 101000846908 Homo sapiens Fc receptor-like protein 5 Proteins 0.000 description 2
- 101000873786 Homo sapiens Glutamate decarboxylase 2 Proteins 0.000 description 2
- 101000819109 Homo sapiens H/ACA ribonucleoprotein complex subunit 1 Proteins 0.000 description 2
- 101001002508 Homo sapiens Immunoglobulin-binding protein 1 Proteins 0.000 description 2
- 101000756759 Homo sapiens MHC class II regulatory factor RFX1 Proteins 0.000 description 2
- 101001052493 Homo sapiens Mitogen-activated protein kinase 1 Proteins 0.000 description 2
- 101000950687 Homo sapiens Mitogen-activated protein kinase 7 Proteins 0.000 description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 2
- 101000829992 Homo sapiens N-acetylglucosamine-6-sulfatase Proteins 0.000 description 2
- 101000633516 Homo sapiens Nuclear receptor subfamily 2 group F member 6 Proteins 0.000 description 2
- 101000606506 Homo sapiens Receptor-type tyrosine-protein phosphatase eta Proteins 0.000 description 2
- 101000633778 Homo sapiens SLAM family member 5 Proteins 0.000 description 2
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 description 2
- 101000633782 Homo sapiens SLAM family member 8 Proteins 0.000 description 2
- 101000633792 Homo sapiens SLAM family member 9 Proteins 0.000 description 2
- 101000800583 Homo sapiens Transcription factor 20 Proteins 0.000 description 2
- 101000638161 Homo sapiens Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 2
- 101000638255 Homo sapiens Tumor necrosis factor ligand superfamily member 8 Proteins 0.000 description 2
- 101000638251 Homo sapiens Tumor necrosis factor ligand superfamily member 9 Proteins 0.000 description 2
- 101000610602 Homo sapiens Tumor necrosis factor receptor superfamily member 10C Proteins 0.000 description 2
- 101000795169 Homo sapiens Tumor necrosis factor receptor superfamily member 13C Proteins 0.000 description 2
- 101000801255 Homo sapiens Tumor necrosis factor receptor superfamily member 17 Proteins 0.000 description 2
- 101000801227 Homo sapiens Tumor necrosis factor receptor superfamily member 19 Proteins 0.000 description 2
- 101000611185 Homo sapiens Tumor necrosis factor receptor superfamily member 5 Proteins 0.000 description 2
- 101000920026 Homo sapiens Tumor necrosis factor receptor superfamily member EDAR Proteins 0.000 description 2
- 102100034980 ICOS ligand Human genes 0.000 description 2
- 101710093458 ICOS ligand Proteins 0.000 description 2
- 108010073807 IgG Receptors Proteins 0.000 description 2
- 102100021042 Immunoglobulin-binding protein 1 Human genes 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102000013264 Interleukin-23 Human genes 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108010029660 Intrinsically Disordered Proteins Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 102100023437 Junctional adhesion molecule-like Human genes 0.000 description 2
- 102100023129 Keratin, type I cytoskeletal 9 Human genes 0.000 description 2
- 108010070514 Keratin-1 Proteins 0.000 description 2
- 108010070585 Keratin-9 Proteins 0.000 description 2
- 102100020880 Kit ligand Human genes 0.000 description 2
- 101710177504 Kit ligand Proteins 0.000 description 2
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- 108010043135 L-methionine gamma-lyase Proteins 0.000 description 2
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 2
- 102100026894 Lymphotoxin-beta Human genes 0.000 description 2
- 108090000362 Lymphotoxin-beta Proteins 0.000 description 2
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 2
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 2
- 102100030352 Membrane-associated phosphatidylinositol transfer protein 2 Human genes 0.000 description 2
- 101710104270 Membrane-associated phosphatidylinositol transfer protein 2 Proteins 0.000 description 2
- 101000783591 Micrurus clarki Clarkitoxin-1 Proteins 0.000 description 2
- 101000963932 Micrurus frontalis Frontoxin II Proteins 0.000 description 2
- 101000783588 Micrurus mipartitus Mipartoxin-1 Proteins 0.000 description 2
- 101000963935 Micrurus nigrocinctus Nicotinic acetylcholine receptor-binding protein Mnn-1A Proteins 0.000 description 2
- 101000964147 Micrurus nigrocinctus Nicotinic acetylcholine receptor-binding protein Mnn-3C Proteins 0.000 description 2
- 101000964140 Micrurus nigrocinctus Nicotinic acetylcholine receptor-binding protein Mnn-4 Proteins 0.000 description 2
- 101000724922 Micrurus pyrrhocryptus Venom protein E2 Proteins 0.000 description 2
- 101000724923 Micrurus surinamensis Short neurotoxin MS11 Proteins 0.000 description 2
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 2
- 102100037805 Mitogen-activated protein kinase 7 Human genes 0.000 description 2
- 101000852213 Mus musculus THO complex subunit 4 Proteins 0.000 description 2
- 101000597780 Mus musculus Tumor necrosis factor ligand superfamily member 18 Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 102100023282 N-acetylglucosamine-6-sulfatase Human genes 0.000 description 2
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 101000724924 Naja kaouthia Nakoroxin Proteins 0.000 description 2
- 102100023996 Neutral ceramidase Human genes 0.000 description 2
- 102100029528 Nuclear receptor subfamily 2 group F member 6 Human genes 0.000 description 2
- 102100022670 Nuclear receptor subfamily 6 group A member 1 Human genes 0.000 description 2
- 102100037589 OX-2 membrane glycoprotein Human genes 0.000 description 2
- 101000963934 Ophiophagus hannah Neurotoxin Oh9-1 Proteins 0.000 description 2
- 101000724920 Ophiophagus hannah Short neurotoxin OH-26 Proteins 0.000 description 2
- 101000963927 Ophiophagus hannah Short neurotoxin OH-32 Proteins 0.000 description 2
- 101000724910 Ophiophagus hannah Short neurotoxin OH-46 Proteins 0.000 description 2
- 101000964138 Ophiophagus hannah Short neurotoxin OH-5 Proteins 0.000 description 2
- 101000724915 Ophiophagus hannah Short neurotoxin SNTX11 Proteins 0.000 description 2
- 101000724916 Ophiophagus hannah Short neurotoxin SNTX14 Proteins 0.000 description 2
- 101000724918 Ophiophagus hannah Short neurotoxin SNTX26 Proteins 0.000 description 2
- 101000724908 Ophiophagus hannah Short neurotoxin SNTX6 Proteins 0.000 description 2
- 101000964146 Ophiophagus hannah Weak neurotoxin WNTX33 Proteins 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 101100242312 Oryza sativa subsp. japonica OSK1 gene Proteins 0.000 description 2
- 101100533605 Oryza sativa subsp. japonica SKP1 gene Proteins 0.000 description 2
- 101000964133 Oxyuranus microlepidotus Toxin 3FTx-Oxy5 Proteins 0.000 description 2
- 101000724919 Oxyuranus scutellatus scutellatus Scutelatoxin Proteins 0.000 description 2
- 101000964145 Oxyuranus scutellatus scutellatus Short neurotoxin 3 Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 2
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 241000269907 Pleuronectes platessa Species 0.000 description 2
- 102100029740 Poliovirus receptor Human genes 0.000 description 2
- 101710094000 Programmed cell death 1 ligand 1 Proteins 0.000 description 2
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- 108010086890 R-cadherin Proteins 0.000 description 2
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 2
- 101710105008 RNA-binding protein Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102100039808 Receptor-type tyrosine-protein phosphatase eta Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 102100028287 Regulator of nonsense transcripts 1 Human genes 0.000 description 2
- 101710172454 Regulator of nonsense transcripts 1 Proteins 0.000 description 2
- 108010030933 Regulatory Factor X1 Proteins 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 102100029216 SLAM family member 5 Human genes 0.000 description 2
- 102100029198 SLAM family member 7 Human genes 0.000 description 2
- 102100029214 SLAM family member 8 Human genes 0.000 description 2
- 102100029196 SLAM family member 9 Human genes 0.000 description 2
- 229940124639 Selective inhibitor Drugs 0.000 description 2
- 102100034136 Serine/threonine-protein kinase receptor R3 Human genes 0.000 description 2
- 101710082813 Serine/threonine-protein kinase receptor R3 Proteins 0.000 description 2
- 102100021913 Sperm-associated antigen 8 Human genes 0.000 description 2
- 101710098579 Sperm-associated antigen 8 Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 101710111844 Synergin gamma Proteins 0.000 description 2
- 102100035936 Syntaxin-2 Human genes 0.000 description 2
- 102100033447 T-lymphocyte surface antigen Ly-9 Human genes 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 2
- 108010000499 Thromboplastin Proteins 0.000 description 2
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 2
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 2
- 102100030859 Tissue factor Human genes 0.000 description 2
- 241000723792 Tobacco etch virus Species 0.000 description 2
- 102100039189 Transcription factor Maf Human genes 0.000 description 2
- 102100030248 Transcription factor SOX-1 Human genes 0.000 description 2
- 101710196852 Transcriptional activator protein Pur-alpha Proteins 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 102100023178 Transcriptional repressor scratch 2 Human genes 0.000 description 2
- 102100024568 Tumor necrosis factor ligand superfamily member 11 Human genes 0.000 description 2
- 101710097155 Tumor necrosis factor ligand superfamily member 12 Proteins 0.000 description 2
- 102100024587 Tumor necrosis factor ligand superfamily member 15 Human genes 0.000 description 2
- 102100026890 Tumor necrosis factor ligand superfamily member 4 Human genes 0.000 description 2
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 2
- 102100040115 Tumor necrosis factor receptor superfamily member 10C Human genes 0.000 description 2
- 102100032236 Tumor necrosis factor receptor superfamily member 11B Human genes 0.000 description 2
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 2
- 102100033760 Tumor necrosis factor receptor superfamily member 19 Human genes 0.000 description 2
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 description 2
- 102100030810 Tumor necrosis factor receptor superfamily member EDAR Human genes 0.000 description 2
- 108010035075 Tyrosine decarboxylase Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 102100020722 WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 Human genes 0.000 description 2
- 102100036021 WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 Human genes 0.000 description 2
- 102100039966 Zinc finger homeobox protein 3 Human genes 0.000 description 2
- 101710180292 Zinc finger homeobox protein 3 Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000009824 affinity maturation Effects 0.000 description 2
- 229960003318 alteplase Drugs 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000003098 androgen Substances 0.000 description 2
- 108010080146 androgen receptors Proteins 0.000 description 2
- 108010072788 angiogenin Proteins 0.000 description 2
- 108010069801 angiopoietin 4 Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 230000000599 auto-anti-genic effect Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229930003827 cannabinoid Natural products 0.000 description 2
- 239000003557 cannabinoid Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000007073 chemical hydrolysis Effects 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 229960004630 chlorambucil Drugs 0.000 description 2
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 108050003126 conotoxin Proteins 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 108050004038 cystatin Proteins 0.000 description 2
- LEVWYRKDKASIDU-IMJSIDKUSA-N cystine group Chemical group C([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- ILKCDNKCNSNFMP-UHFFFAOYSA-N dimethyl octanediimidate;hydron;dichloride Chemical compound Cl.Cl.COC(=N)CCCCCCC(=N)OC ILKCDNKCNSNFMP-UHFFFAOYSA-N 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- VQNATVDKACXKTF-XELLLNAOSA-N duocarmycin Chemical compound COC1=C(OC)C(OC)=C2NC(C(=O)N3C4=CC(=O)C5=C([C@@]64C[C@@H]6C3)C=C(N5)C(=O)OC)=CC2=C1 VQNATVDKACXKTF-XELLLNAOSA-N 0.000 description 2
- 229960005501 duocarmycin Drugs 0.000 description 2
- 229930184221 duocarmycin Natural products 0.000 description 2
- 150000002066 eicosanoids Chemical class 0.000 description 2
- 230000005686 electrostatic field Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 108700005856 engrailed 2 Proteins 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 229960002949 fluorouracil Drugs 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- CHPZKNULDCNCBW-UHFFFAOYSA-N gallium nitrate Chemical compound [Ga+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O CHPZKNULDCNCBW-UHFFFAOYSA-N 0.000 description 2
- 238000012248 genetic selection Methods 0.000 description 2
- 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 description 2
- 229960004666 glucagon Drugs 0.000 description 2
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 150000002333 glycines Chemical class 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 229960001101 ifosfamide Drugs 0.000 description 2
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001524 infective effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 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 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- GURKHSYORGJETM-WAQYZQTGSA-N irinotecan hydrochloride (anhydrous) Chemical compound Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 GURKHSYORGJETM-WAQYZQTGSA-N 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- YOBAEOGBNPPUQV-UHFFFAOYSA-N iron;trihydrate Chemical compound O.O.O.[Fe].[Fe] YOBAEOGBNPPUQV-UHFFFAOYSA-N 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 229960002486 laronidase Drugs 0.000 description 2
- 108020001756 ligand binding domains Proteins 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229960001428 mercaptopurine Drugs 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229960001156 mitoxantrone Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 229940099216 oncaspar Drugs 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 2
- 108010001564 pegaspargase Proteins 0.000 description 2
- 108091005706 peripheral membrane proteins Proteins 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 108010094020 polyglycine Proteins 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000002818 protein evolution Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 2
- 229960004622 raloxifene Drugs 0.000 description 2
- 239000000018 receptor agonist Substances 0.000 description 2
- 229940044601 receptor agonist Drugs 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 108010039827 snRNP Core Proteins Proteins 0.000 description 2
- 239000003998 snake venom Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- PVYJZLYGTZKPJE-UHFFFAOYSA-N streptonigrin Chemical compound C=1C=C2C(=O)C(OC)=C(N)C(=O)C2=NC=1C(C=1N)=NC(C(O)=O)=C(C)C=1C1=CC=C(OC)C(OC)=C1O PVYJZLYGTZKPJE-UHFFFAOYSA-N 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000003774 sulfhydryl reagent Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 229960001196 thiotepa Drugs 0.000 description 2
- 150000003595 thromboxanes Chemical class 0.000 description 2
- 229960003087 tioguanine Drugs 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 238000000101 transmission high energy electron diffraction Methods 0.000 description 2
- 238000005829 trimerization reaction Methods 0.000 description 2
- 229910052720 vanadium Inorganic materials 0.000 description 2
- 229960003048 vinblastine Drugs 0.000 description 2
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 2
- 229960004528 vincristine Drugs 0.000 description 2
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 description 1
- FLEHQRTTWKDNGI-XTJILODYSA-N (1s,3r)-5-[(2e)-2-[(7ar)-1-[(2s)-5-(cyclopropylamino)pentan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-2-methylidenecyclohexane-1,3-diol Chemical compound C([C@H](C)C1[C@]2(CCCC(/C2CC1)=C\C=C1C[C@@H](O)C(=C)[C@@H](O)C1)C)CCNC1CC1 FLEHQRTTWKDNGI-XTJILODYSA-N 0.000 description 1
- TYKASZBHFXBROF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(2,5-dioxopyrrol-1-yl)acetate Chemical compound O=C1CCC(=O)N1OC(=O)CN1C(=O)C=CC1=O TYKASZBHFXBROF-UHFFFAOYSA-N 0.000 description 1
- XUDGDVPXDYGCTG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxyethylsulfonyl]ethyl carbonate Chemical compound O=C1CCC(=O)N1OC(=O)OCCS(=O)(=O)CCOC(=O)ON1C(=O)CCC1=O XUDGDVPXDYGCTG-UHFFFAOYSA-N 0.000 description 1
- JKHVDAUOODACDU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCN1C(=O)C=CC1=O JKHVDAUOODACDU-UHFFFAOYSA-N 0.000 description 1
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- FXYPGCIGRDZWNR-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)CCC1=O FXYPGCIGRDZWNR-UHFFFAOYSA-N 0.000 description 1
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- BQWBEDSJTMWJAE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[(2-iodoacetyl)amino]benzoate Chemical compound C1=CC(NC(=O)CI)=CC=C1C(=O)ON1C(=O)CCC1=O BQWBEDSJTMWJAE-UHFFFAOYSA-N 0.000 description 1
- GKSPIZSKQWTXQG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate Chemical compound C=1C=C(C(=O)ON2C(CCC2=O)=O)C=CC=1C(C)SSC1=CC=CC=N1 GKSPIZSKQWTXQG-UHFFFAOYSA-N 0.000 description 1
- PMJWDPGOWBRILU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCC(C=C1)=CC=C1N1C(=O)C=CC1=O PMJWDPGOWBRILU-UHFFFAOYSA-N 0.000 description 1
- RBAFCMJBDZWZIV-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-azido-2-hydroxybenzoate Chemical compound OC1=CC(N=[N+]=[N-])=CC=C1C(=O)ON1C(=O)CCC1=O RBAFCMJBDZWZIV-UHFFFAOYSA-N 0.000 description 1
- FUOJEDZPVVDXHI-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 5-azido-2-nitrobenzoate Chemical compound [O-][N+](=O)C1=CC=C(N=[N+]=[N-])C=C1C(=O)ON1C(=O)CCC1=O FUOJEDZPVVDXHI-UHFFFAOYSA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 1
- NGXDNMNOQDVTRL-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(4-azido-2-nitroanilino)hexanoate Chemical compound [O-][N+](=O)C1=CC(N=[N+]=[N-])=CC=C1NCCCCCC(=O)ON1C(=O)CCC1=O NGXDNMNOQDVTRL-UHFFFAOYSA-N 0.000 description 1
- WCMOHMXWOOBVMZ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCN1C(=O)C=CC1=O WCMOHMXWOOBVMZ-UHFFFAOYSA-N 0.000 description 1
- QYEAAMBIUQLHFQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 QYEAAMBIUQLHFQ-UHFFFAOYSA-N 0.000 description 1
- IHVODYOQUSEYJJ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]amino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)C(CC1)CCC1CN1C(=O)C=CC1=O IHVODYOQUSEYJJ-UHFFFAOYSA-N 0.000 description 1
- WDQLRUYAYXDIFW-RWKIJVEZSA-N (2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-4-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 WDQLRUYAYXDIFW-RWKIJVEZSA-N 0.000 description 1
- FLWWDYNPWOSLEO-HQVZTVAUSA-N (2s)-2-[[4-[1-(2-amino-4-oxo-1h-pteridin-6-yl)ethyl-methylamino]benzoyl]amino]pentanedioic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1C(C)N(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FLWWDYNPWOSLEO-HQVZTVAUSA-N 0.000 description 1
- RRUYWEMUWIRRNB-LURJTMIESA-N (2s)-6-amino-2-[carboxy(methyl)amino]hexanoic acid Chemical compound OC(=O)N(C)[C@H](C(O)=O)CCCCN RRUYWEMUWIRRNB-LURJTMIESA-N 0.000 description 1
- CGMTUJFWROPELF-YPAAEMCBSA-N (3E,5S)-5-[(2S)-butan-2-yl]-3-(1-hydroxyethylidene)pyrrolidine-2,4-dione Chemical compound CC[C@H](C)[C@@H]1NC(=O)\C(=C(/C)O)C1=O CGMTUJFWROPELF-YPAAEMCBSA-N 0.000 description 1
- LTDQGCFMTVHZKP-UHFFFAOYSA-N (4-bromophenyl)-(4,6-dimethoxy-3-methyl-1-benzofuran-2-yl)methanone Chemical compound O1C2=CC(OC)=CC(OC)=C2C(C)=C1C(=O)C1=CC=C(Br)C=C1 LTDQGCFMTVHZKP-UHFFFAOYSA-N 0.000 description 1
- XRBSKUSTLXISAB-XVVDYKMHSA-N (5r,6r,7r,8r)-8-hydroxy-7-(hydroxymethyl)-5-(3,4,5-trimethoxyphenyl)-5,6,7,8-tetrahydrobenzo[f][1,3]benzodioxole-6-carboxylic acid Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@H](O)[C@@H](CO)[C@@H]2C(O)=O)=C1 XRBSKUSTLXISAB-XVVDYKMHSA-N 0.000 description 1
- XRBSKUSTLXISAB-UHFFFAOYSA-N (7R,7'R,8R,8'R)-form-Podophyllic acid Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C(CO)C2C(O)=O)=C1 XRBSKUSTLXISAB-UHFFFAOYSA-N 0.000 description 1
- AESVUZLWRXEGEX-DKCAWCKPSA-N (7S,9R)-7-[(2S,4R,5R,6R)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione iron(3+) Chemical compound [Fe+3].COc1cccc2C(=O)c3c(O)c4C[C@@](O)(C[C@H](O[C@@H]5C[C@@H](N)[C@@H](O)[C@@H](C)O5)c4c(O)c3C(=O)c12)C(=O)CO AESVUZLWRXEGEX-DKCAWCKPSA-N 0.000 description 1
- JXVAMODRWBNUSF-KZQKBALLSA-N (7s,9r,10r)-7-[(2r,4s,5s,6s)-5-[[(2s,4as,5as,7s,9s,9ar,10ar)-2,9-dimethyl-3-oxo-4,4a,5a,6,7,9,9a,10a-octahydrodipyrano[4,2-a:4',3'-e][1,4]dioxin-7-yl]oxy]-4-(dimethylamino)-6-methyloxan-2-yl]oxy-10-[(2s,4s,5s,6s)-4-(dimethylamino)-5-hydroxy-6-methyloxan-2 Chemical compound O([C@@H]1C2=C(O)C=3C(=O)C4=CC=CC(O)=C4C(=O)C=3C(O)=C2[C@@H](O[C@@H]2O[C@@H](C)[C@@H](O[C@@H]3O[C@@H](C)[C@H]4O[C@@H]5O[C@@H](C)C(=O)C[C@@H]5O[C@H]4C3)[C@H](C2)N(C)C)C[C@]1(O)CC)[C@H]1C[C@H](N(C)C)[C@H](O)[C@H](C)O1 JXVAMODRWBNUSF-KZQKBALLSA-N 0.000 description 1
- FPVKHBSQESCIEP-UHFFFAOYSA-N (8S)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol Natural products C1C(O)C(CO)OC1N1C(NC=NCC2O)=C2N=C1 FPVKHBSQESCIEP-UHFFFAOYSA-N 0.000 description 1
- IEXUMDBQLIVNHZ-YOUGDJEHSA-N (8s,11r,13r,14s,17s)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(3-hydroxypropyl)-13-methyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one Chemical compound C1=CC(N(C)C)=CC=C1[C@@H]1C2=C3CCC(=O)C=C3CC[C@H]2[C@H](CC[C@]2(O)CCCO)[C@@]2(C)C1 IEXUMDBQLIVNHZ-YOUGDJEHSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- LKJPYSCBVHEWIU-KRWDZBQOSA-N (R)-bicalutamide Chemical compound C([C@@](O)(C)C(=O)NC=1C=C(C(C#N)=CC=1)C(F)(F)F)S(=O)(=O)C1=CC=C(F)C=C1 LKJPYSCBVHEWIU-KRWDZBQOSA-N 0.000 description 1
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (e)-1-[(2s)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 1
- JXSSPZOCHGZWJP-UHFFFAOYSA-N 1,2-difluoro-3,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C(F)=C1[N+]([O-])=O JXSSPZOCHGZWJP-UHFFFAOYSA-N 0.000 description 1
- SGVWDRVQIYUSRA-UHFFFAOYSA-N 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CCSSCCN1C(=O)C=CC1=O SGVWDRVQIYUSRA-UHFFFAOYSA-N 0.000 description 1
- DIYPCWKHSODVAP-UHFFFAOYSA-N 1-[3-(2,5-dioxopyrrol-1-yl)benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 DIYPCWKHSODVAP-UHFFFAOYSA-N 0.000 description 1
- XKSOTQXTPALQMY-UHFFFAOYSA-N 1-[3-[(4-azidophenyl)disulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCSSC1=CC=C(N=[N+]=[N-])C=C1 XKSOTQXTPALQMY-UHFFFAOYSA-N 0.000 description 1
- XKQYCEFPFNDDSJ-UHFFFAOYSA-N 1-[3-[2-[(4-azido-2-hydroxybenzoyl)amino]ethyldisulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound OC1=CC(N=[N+]=[N-])=CC=C1C(=O)NCCSSCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O XKQYCEFPFNDDSJ-UHFFFAOYSA-N 0.000 description 1
- VOTJUWBJENROFB-UHFFFAOYSA-N 1-[3-[[3-(2,5-dioxo-3-sulfopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)C(S(O)(=O)=O)CC1=O VOTJUWBJENROFB-UHFFFAOYSA-N 0.000 description 1
- CULQNACJHGHAER-UHFFFAOYSA-N 1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 CULQNACJHGHAER-UHFFFAOYSA-N 0.000 description 1
- UPNUQQDXHCUWSG-UHFFFAOYSA-N 1-[6-(4-azido-2-nitroanilino)hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC1=CC=C(N=[N+]=[N-])C=C1[N+]([O-])=O UPNUQQDXHCUWSG-UHFFFAOYSA-N 0.000 description 1
- LEBVLXFERQHONN-UHFFFAOYSA-N 1-butyl-N-(2,6-dimethylphenyl)piperidine-2-carboxamide Chemical compound CCCCN1CCCCC1C(=O)NC1=C(C)C=CC=C1C LEBVLXFERQHONN-UHFFFAOYSA-N 0.000 description 1
- NQTSTBMCCAVWOS-UHFFFAOYSA-N 1-dimethoxyphosphoryl-3-phenoxypropan-2-one Chemical compound COP(=O)(OC)CC(=O)COC1=CC=CC=C1 NQTSTBMCCAVWOS-UHFFFAOYSA-N 0.000 description 1
- KKVYYGGCHJGEFJ-UHFFFAOYSA-N 1-n-(4-chlorophenyl)-6-methyl-5-n-[3-(7h-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine Chemical compound N=1C=CC2=C(NC=3C(=CC=CN=3)C=3C=4N=CNC=4N=CN=3)C(C)=CC=C2C=1NC1=CC=C(Cl)C=C1 KKVYYGGCHJGEFJ-UHFFFAOYSA-N 0.000 description 1
- 102000004899 14-3-3 Proteins Human genes 0.000 description 1
- BTOTXLJHDSNXMW-POYBYMJQSA-N 2,3-dideoxyuridine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(=O)NC(=O)C=C1 BTOTXLJHDSNXMW-POYBYMJQSA-N 0.000 description 1
- BOMZMNZEXMAQQW-UHFFFAOYSA-N 2,5,11-trimethyl-6h-pyrido[4,3-b]carbazol-2-ium-9-ol;acetate Chemical compound CC([O-])=O.C[N+]1=CC=C2C(C)=C(NC=3C4=CC(O)=CC=3)C4=C(C)C2=C1 BOMZMNZEXMAQQW-UHFFFAOYSA-N 0.000 description 1
- ASNTZYQMIUCEBV-UHFFFAOYSA-N 2,5-dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 ASNTZYQMIUCEBV-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- QZDDFQLIQRYMBV-UHFFFAOYSA-N 2-[3-nitro-2-(2-nitrophenyl)-4-oxochromen-8-yl]acetic acid Chemical compound OC(=O)CC1=CC=CC(C(C=2[N+]([O-])=O)=O)=C1OC=2C1=CC=CC=C1[N+]([O-])=O QZDDFQLIQRYMBV-UHFFFAOYSA-N 0.000 description 1
- QCXJFISCRQIYID-IAEPZHFASA-N 2-amino-1-n-[(3s,6s,7r,10s,16s)-3-[(2s)-butan-2-yl]-7,11,14-trimethyl-2,5,9,12,15-pentaoxo-10-propan-2-yl-8-oxa-1,4,11,14-tetrazabicyclo[14.3.0]nonadecan-6-yl]-4,6-dimethyl-3-oxo-9-n-[(3s,6s,7r,10s,16s)-7,11,14-trimethyl-2,5,9,12,15-pentaoxo-3,10-di(propa Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N=C2C(C(=O)N[C@@H]3C(=O)N[C@H](C(N4CCC[C@H]4C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]3C)=O)[C@@H](C)CC)=C(N)C(=O)C(C)=C2O2)C2=C(C)C=C1 QCXJFISCRQIYID-IAEPZHFASA-N 0.000 description 1
- FDAYLTPAFBGXAB-UHFFFAOYSA-N 2-chloro-n,n-bis(2-chloroethyl)ethanamine Chemical compound ClCCN(CCCl)CCCl FDAYLTPAFBGXAB-UHFFFAOYSA-N 0.000 description 1
- VNBAOSVONFJBKP-UHFFFAOYSA-N 2-chloro-n,n-bis(2-chloroethyl)propan-1-amine;hydrochloride Chemical compound Cl.CC(Cl)CN(CCCl)CCCl VNBAOSVONFJBKP-UHFFFAOYSA-N 0.000 description 1
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- NDMPLJNOPCLANR-UHFFFAOYSA-N 3,4-dihydroxy-15-(4-hydroxy-18-methoxycarbonyl-5,18-seco-ibogamin-18-yl)-16-methoxy-1-methyl-6,7-didehydro-aspidospermidine-3-carboxylic acid methyl ester Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 NDMPLJNOPCLANR-UHFFFAOYSA-N 0.000 description 1
- PWMYMKOUNYTVQN-UHFFFAOYSA-N 3-(8,8-diethyl-2-aza-8-germaspiro[4.5]decan-2-yl)-n,n-dimethylpropan-1-amine Chemical compound C1C[Ge](CC)(CC)CCC11CN(CCCN(C)C)CC1 PWMYMKOUNYTVQN-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- JMUAKWNHKQBPGJ-UHFFFAOYSA-N 3-(pyridin-2-yldisulfanyl)-n-[4-[3-(pyridin-2-yldisulfanyl)propanoylamino]butyl]propanamide Chemical compound C=1C=CC=NC=1SSCCC(=O)NCCCCNC(=O)CCSSC1=CC=CC=N1 JMUAKWNHKQBPGJ-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- DODQJNMQWMSYGS-QPLCGJKRSA-N 4-[(z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-phenylbut-1-en-2-yl]phenol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 DODQJNMQWMSYGS-QPLCGJKRSA-N 0.000 description 1
- SSMIFVHARFVINF-UHFFFAOYSA-N 4-amino-1,8-naphthalimide Chemical compound O=C1NC(=O)C2=CC=CC3=C2C1=CC=C3N SSMIFVHARFVINF-UHFFFAOYSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 description 1
- QLHLYJHNOCILIT-UHFFFAOYSA-N 4-o-(2,5-dioxopyrrolidin-1-yl) 1-o-[2-[4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxobutanoyl]oxyethyl] butanedioate Chemical compound O=C1CCC(=O)N1OC(=O)CCC(=O)OCCOC(=O)CCC(=O)ON1C(=O)CCC1=O QLHLYJHNOCILIT-UHFFFAOYSA-N 0.000 description 1
- 102100038049 5'-AMP-activated protein kinase subunit beta-2 Human genes 0.000 description 1
- IDPUKCWIGUEADI-UHFFFAOYSA-N 5-[bis(2-chloroethyl)amino]uracil Chemical compound ClCCN(CCCl)C1=CNC(=O)NC1=O IDPUKCWIGUEADI-UHFFFAOYSA-N 0.000 description 1
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 1
- WYXSYVWAUAUWLD-SHUUEZRQSA-N 6-azauridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=N1 WYXSYVWAUAUWLD-SHUUEZRQSA-N 0.000 description 1
- 229960005538 6-diazo-5-oxo-L-norleucine Drugs 0.000 description 1
- YCWQAMGASJSUIP-YFKPBYRVSA-N 6-diazo-5-oxo-L-norleucine Chemical compound OC(=O)[C@@H](N)CCC(=O)C=[N+]=[N-] YCWQAMGASJSUIP-YFKPBYRVSA-N 0.000 description 1
- ULGJWNIHLSLQPZ-UHFFFAOYSA-N 7-[(6,8-dichloro-1,2,3,4-tetrahydroacridin-9-yl)amino]-n-[2-(1h-indol-3-yl)ethyl]heptanamide Chemical compound C1CCCC2=NC3=CC(Cl)=CC(Cl)=C3C(NCCCCCCC(=O)NCCC=3C4=CC=CC=C4NC=3)=C21 ULGJWNIHLSLQPZ-UHFFFAOYSA-N 0.000 description 1
- HCAJQHYUCKICQH-VPENINKCSA-N 8-Oxo-7,8-dihydro-2'-deoxyguanosine Chemical compound C1=2NC(N)=NC(=O)C=2NC(=O)N1[C@H]1C[C@H](O)[C@@H](CO)O1 HCAJQHYUCKICQH-VPENINKCSA-N 0.000 description 1
- RTAPDZBZLSXHQQ-UHFFFAOYSA-N 8-methyl-3,7-dihydropurine-2,6-dione Chemical class N1C(=O)NC(=O)C2=C1N=C(C)N2 RTAPDZBZLSXHQQ-UHFFFAOYSA-N 0.000 description 1
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 description 1
- HDZZVAMISRMYHH-UHFFFAOYSA-N 9beta-Ribofuranosyl-7-deazaadenin Natural products C1=CC=2C(N)=NC=NC=2N1C1OC(CO)C(O)C1O HDZZVAMISRMYHH-UHFFFAOYSA-N 0.000 description 1
- 102100027398 A disintegrin and metalloproteinase with thrombospondin motifs 1 Human genes 0.000 description 1
- 102100027400 A disintegrin and metalloproteinase with thrombospondin motifs 4 Human genes 0.000 description 1
- 108091022885 ADAM Proteins 0.000 description 1
- 102000029791 ADAM Human genes 0.000 description 1
- 108091007504 ADAM10 Proteins 0.000 description 1
- 108091007507 ADAM12 Proteins 0.000 description 1
- 108091007505 ADAM17 Proteins 0.000 description 1
- 101710188409 ADAMTS-like protein 1 Proteins 0.000 description 1
- 108091005660 ADAMTS1 Proteins 0.000 description 1
- 108091005664 ADAMTS4 Proteins 0.000 description 1
- 108091005663 ADAMTS5 Proteins 0.000 description 1
- 102000051389 ADAMTS5 Human genes 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- 101150107888 AKT2 gene Proteins 0.000 description 1
- 101150104383 ALOX5AP gene Proteins 0.000 description 1
- 102000000872 ATM Human genes 0.000 description 1
- 102100027938 ATR-interacting protein Human genes 0.000 description 1
- 101001082110 Acanthamoeba polyphaga mimivirus Eukaryotic translation initiation factor 4E homolog Proteins 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 102100034111 Activin receptor type-1 Human genes 0.000 description 1
- 102100034134 Activin receptor type-1B Human genes 0.000 description 1
- 101710173011 Activin receptor type-1B Proteins 0.000 description 1
- 102100034135 Activin receptor type-1C Human genes 0.000 description 1
- 101710173005 Activin receptor type-1C Proteins 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- ORILYTVJVMAKLC-UHFFFAOYSA-N Adamantane Natural products C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 1
- 102100026423 Adhesion G protein-coupled receptor E5 Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100036601 Aggrecan core protein Human genes 0.000 description 1
- 108010067219 Aggrecans Proteins 0.000 description 1
- 101150051155 Akt3 gene Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 102100033312 Alpha-2-macroglobulin Human genes 0.000 description 1
- CEIZFXOZIQNICU-UHFFFAOYSA-N Alternaria alternata Crofton-weed toxin Natural products CCC(C)C1NC(=O)C(C(C)=O)=C1O CEIZFXOZIQNICU-UHFFFAOYSA-N 0.000 description 1
- 102100040409 Ameloblastin Human genes 0.000 description 1
- 101710081264 Ameloblastin Proteins 0.000 description 1
- 102100025981 Aminoacylase-1 Human genes 0.000 description 1
- 102000000782 Aminopeptidase A Human genes 0.000 description 1
- 101710190488 Aminopeptidase P1 Proteins 0.000 description 1
- 101710190490 Aminopeptidase P2 Proteins 0.000 description 1
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- 102000004400 Aminopeptidases Human genes 0.000 description 1
- 102100032040 Amphoterin-induced protein 2 Human genes 0.000 description 1
- 102100031993 Amphoterin-induced protein 3 Human genes 0.000 description 1
- 101710168919 Amyloid beta precursor like protein 1 Proteins 0.000 description 1
- 102100040055 Amyloid beta precursor like protein 1 Human genes 0.000 description 1
- 102100040038 Amyloid beta precursor like protein 2 Human genes 0.000 description 1
- 101710168921 Amyloid beta precursor like protein 2 Proteins 0.000 description 1
- 101000654311 Androctonus australis Alpha-mammal toxin AaH2 Proteins 0.000 description 1
- 108010048154 Angiopoietin-1 Proteins 0.000 description 1
- 102100034594 Angiopoietin-1 Human genes 0.000 description 1
- 102100034608 Angiopoietin-2 Human genes 0.000 description 1
- 108010048036 Angiopoietin-2 Proteins 0.000 description 1
- 102100033402 Angiopoietin-4 Human genes 0.000 description 1
- 102100025672 Angiopoietin-related protein 2 Human genes 0.000 description 1
- 102100025668 Angiopoietin-related protein 3 Human genes 0.000 description 1
- 102100025674 Angiopoietin-related protein 4 Human genes 0.000 description 1
- 102400000068 Angiostatin Human genes 0.000 description 1
- 108010079709 Angiostatins Proteins 0.000 description 1
- 101710185050 Angiotensin-converting enzyme Proteins 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 102000017620 Annexin A10 Human genes 0.000 description 1
- 108050005848 Annexin A10 Proteins 0.000 description 1
- 102100034273 Annexin A7 Human genes 0.000 description 1
- 108010039940 Annexin A7 Proteins 0.000 description 1
- 102100031325 Anthrax toxin receptor 2 Human genes 0.000 description 1
- 101710126338 Apamin Proteins 0.000 description 1
- 108010059886 Apolipoprotein A-I Proteins 0.000 description 1
- 102000005666 Apolipoprotein A-I Human genes 0.000 description 1
- 101710095342 Apolipoprotein B Proteins 0.000 description 1
- 102100040202 Apolipoprotein B-100 Human genes 0.000 description 1
- 102100029647 Apoptosis-associated speck-like protein containing a CARD Human genes 0.000 description 1
- 108010062544 Apoptotic Protease-Activating Factor 1 Proteins 0.000 description 1
- 102100034524 Apoptotic protease-activating factor 1 Human genes 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101000838034 Arabidopsis thaliana Transcription initiation factor TFIID subunit 15 Proteins 0.000 description 1
- 102100029361 Aromatase Human genes 0.000 description 1
- 108010078554 Aromatase Proteins 0.000 description 1
- 102100026376 Artemin Human genes 0.000 description 1
- 101710205806 Artemin Proteins 0.000 description 1
- 102100026292 Asialoglycoprotein receptor 1 Human genes 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 102100037293 Atrial natriuretic peptide-converting enzyme Human genes 0.000 description 1
- 101710133555 Atrial natriuretic peptide-converting enzyme Proteins 0.000 description 1
- 102100035682 Axin-1 Human genes 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical class C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 102100025218 B-cell differentiation antigen CD72 Human genes 0.000 description 1
- 101710109862 B-cell lymphoma/leukemia 10 Proteins 0.000 description 1
- 102100037598 B-cell lymphoma/leukemia 10 Human genes 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 101150033765 BAG1 gene Proteins 0.000 description 1
- 101700002522 BARD1 Proteins 0.000 description 1
- PCLCDPVEEFVAAQ-UHFFFAOYSA-N BCA 1 Chemical compound CC(CO)CCCC(C)C1=CCC(C)(O)C1CC2=C(O)C(O)CCC2=O PCLCDPVEEFVAAQ-UHFFFAOYSA-N 0.000 description 1
- 102000004276 BCL2-related protein A1 Human genes 0.000 description 1
- 108090000879 BCL2-related protein A1 Proteins 0.000 description 1
- 102100037140 BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Human genes 0.000 description 1
- 102100025440 BMP-binding endothelial regulator protein Human genes 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 102100028048 BRCA1-associated RING domain protein 1 Human genes 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 1
- 108010064528 Basigin Proteins 0.000 description 1
- 102000015279 Basigin Human genes 0.000 description 1
- 102100032412 Basigin Human genes 0.000 description 1
- 102000001765 Bcl-2-Like Protein 11 Human genes 0.000 description 1
- 108010040168 Bcl-2-Like Protein 11 Proteins 0.000 description 1
- 102100026596 Bcl-2-like protein 1 Human genes 0.000 description 1
- 102100022541 Bcl-2-related ovarian killer protein Human genes 0.000 description 1
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 1
- 102100026349 Beta-1,4-galactosyltransferase 1 Human genes 0.000 description 1
- 102100031006 Beta-Ala-His dipeptidase Human genes 0.000 description 1
- 101710192841 Beta-Ala-His dipeptidase Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 101710125298 Beta-defensin 2 Proteins 0.000 description 1
- 102100038326 Beta-defensin 4A Human genes 0.000 description 1
- 101710176951 Beta-defensin 4A Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 1
- 101800001382 Betacellulin Proteins 0.000 description 1
- 102000004954 Biglycan Human genes 0.000 description 1
- 108090001138 Biglycan Proteins 0.000 description 1
- 102100023109 Bile acyl-CoA synthetase Human genes 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 1
- 108010049951 Bone Morphogenetic Protein 3 Proteins 0.000 description 1
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 1
- 108010049976 Bone Morphogenetic Protein 5 Proteins 0.000 description 1
- 108010049974 Bone Morphogenetic Protein 6 Proteins 0.000 description 1
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 1
- 108010040422 Bone Morphogenetic Protein Receptors Proteins 0.000 description 1
- 102000001893 Bone Morphogenetic Protein Receptors Human genes 0.000 description 1
- 102100028726 Bone morphogenetic protein 10 Human genes 0.000 description 1
- 101710118482 Bone morphogenetic protein 10 Proteins 0.000 description 1
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 1
- 102100024504 Bone morphogenetic protein 3 Human genes 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 102100022526 Bone morphogenetic protein 5 Human genes 0.000 description 1
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 description 1
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 1
- 102100022545 Bone morphogenetic protein 8B Human genes 0.000 description 1
- 102100025423 Bone morphogenetic protein receptor type-1A Human genes 0.000 description 1
- 101710120270 Bone morphogenetic protein receptor type-1A Proteins 0.000 description 1
- 101710120271 Bone morphogenetic protein receptor type-1B Proteins 0.000 description 1
- 102100025422 Bone morphogenetic protein receptor type-2 Human genes 0.000 description 1
- 108050008407 Bone morphogenetic protein receptor type-2 Proteins 0.000 description 1
- 101800004538 Bradykinin Proteins 0.000 description 1
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 102100025401 Breast cancer type 1 susceptibility protein Human genes 0.000 description 1
- 102100025399 Breast cancer type 2 susceptibility protein Human genes 0.000 description 1
- 102100022595 Broad substrate specificity ATP-binding cassette transporter ABCG2 Human genes 0.000 description 1
- 108091005575 Bromodomain-containing proteins Proteins 0.000 description 1
- 102000001805 Bromodomains Human genes 0.000 description 1
- 102100025441 Brother of CDO Human genes 0.000 description 1
- 101710109559 Bucain Proteins 0.000 description 1
- 101710152682 Bucandin Proteins 0.000 description 1
- 241001453380 Burkholderia Species 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 101000685088 Buthus occitanus tunetanus Alpha-toxin Bot1 Proteins 0.000 description 1
- 101000654313 Buthus occitanus tunetanus Neurotoxin Bot2 Proteins 0.000 description 1
- 102100031172 C-C chemokine receptor type 1 Human genes 0.000 description 1
- 101710149814 C-C chemokine receptor type 1 Proteins 0.000 description 1
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 description 1
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 description 1
- 102100024167 C-C chemokine receptor type 3 Human genes 0.000 description 1
- 101710149862 C-C chemokine receptor type 3 Proteins 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 1
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 1
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 1
- 102100036305 C-C chemokine receptor type 8 Human genes 0.000 description 1
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 description 1
- 102100023702 C-C motif chemokine 13 Human genes 0.000 description 1
- 102100023705 C-C motif chemokine 14 Human genes 0.000 description 1
- 102100023703 C-C motif chemokine 15 Human genes 0.000 description 1
- 102100023700 C-C motif chemokine 16 Human genes 0.000 description 1
- 102100023701 C-C motif chemokine 18 Human genes 0.000 description 1
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 1
- 101710112538 C-C motif chemokine 27 Proteins 0.000 description 1
- 102100021942 C-C motif chemokine 28 Human genes 0.000 description 1
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 1
- 102100032366 C-C motif chemokine 7 Human genes 0.000 description 1
- 108010074051 C-Reactive Protein Proteins 0.000 description 1
- 102100028990 C-X-C chemokine receptor type 3 Human genes 0.000 description 1
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 1
- 102100031658 C-X-C chemokine receptor type 5 Human genes 0.000 description 1
- 102100025618 C-X-C chemokine receptor type 6 Human genes 0.000 description 1
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 1
- 102100025279 C-X-C motif chemokine 11 Human genes 0.000 description 1
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 description 1
- 101710085504 C-X-C motif chemokine 6 Proteins 0.000 description 1
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 description 1
- 102100032557 C-type lectin domain family 1 member A Human genes 0.000 description 1
- 101710160443 C-type lectin domain family 1 member A Proteins 0.000 description 1
- 102100032529 C-type lectin domain family 1 member B Human genes 0.000 description 1
- 101710160442 C-type lectin domain family 1 member B Proteins 0.000 description 1
- 102100028668 C-type lectin domain family 4 member C Human genes 0.000 description 1
- 102100028672 C-type lectin domain family 4 member D Human genes 0.000 description 1
- 101710125370 C-type lectin domain family 6 member A Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- YBTVCUSZMGXJNW-FDNBCEJMSA-N C1=C2OCOC2=CC(/C=C\2/C(=C/C=3C=C4OCOC4=CC=3)/COC/2=O)=C1 Chemical compound C1=C2OCOC2=CC(/C=C\2/C(=C/C=3C=C4OCOC4=CC=3)/COC/2=O)=C1 YBTVCUSZMGXJNW-FDNBCEJMSA-N 0.000 description 1
- WMQLLTKSISGWHQ-UHFFFAOYSA-N C1CC(NC(=O)NC)CCC1CCN1CCN(C=2C(=C(Cl)C=CC=2)Cl)CC1 Chemical compound C1CC(NC(=O)NC)CCC1CCN1CCN(C=2C(=C(Cl)C=CC=2)Cl)CC1 WMQLLTKSISGWHQ-UHFFFAOYSA-N 0.000 description 1
- 108700012439 CA9 Proteins 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 102100025752 CASP8 and FADD-like apoptosis regulator Human genes 0.000 description 1
- 101150042405 CCN1 gene Proteins 0.000 description 1
- 102100031023 CCR4-NOT transcription complex subunit 11 Human genes 0.000 description 1
- 102100032976 CCR4-NOT transcription complex subunit 6 Human genes 0.000 description 1
- 102100035893 CD151 antigen Human genes 0.000 description 1
- 108010009992 CD163 antigen Proteins 0.000 description 1
- 102100038077 CD226 antigen Human genes 0.000 description 1
- 108010046080 CD27 Ligand Proteins 0.000 description 1
- 102100025240 CD320 antigen Human genes 0.000 description 1
- 102000049320 CD36 Human genes 0.000 description 1
- 108010045374 CD36 Antigens Proteins 0.000 description 1
- 108010038940 CD48 Antigen Proteins 0.000 description 1
- 102100026862 CD5 antigen-like Human genes 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 102100035793 CD83 antigen Human genes 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- 101710188751 COUP transcription factor 1 Proteins 0.000 description 1
- 101710188750 COUP transcription factor 2 Proteins 0.000 description 1
- 102100035350 CUB domain-containing protein 1 Human genes 0.000 description 1
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 1
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 1
- 101150093802 CXCL1 gene Proteins 0.000 description 1
- 102100024155 Cadherin-11 Human genes 0.000 description 1
- 101710196882 Cadherin-12 Proteins 0.000 description 1
- 102100024156 Cadherin-12 Human genes 0.000 description 1
- 102000014818 Cadherin-13 Human genes 0.000 description 1
- 101710196881 Cadherin-17 Proteins 0.000 description 1
- 102100024152 Cadherin-17 Human genes 0.000 description 1
- 101710196896 Cadherin-22 Proteins 0.000 description 1
- 102100022481 Cadherin-22 Human genes 0.000 description 1
- 102100029756 Cadherin-6 Human genes 0.000 description 1
- 101710097574 Cadherin-8 Proteins 0.000 description 1
- 102100025331 Cadherin-8 Human genes 0.000 description 1
- 101001059668 Caenorhabditis elegans Protein madd-4 Proteins 0.000 description 1
- 101100326430 Caenorhabditis elegans bub-1 gene Proteins 0.000 description 1
- 101100220616 Caenorhabditis elegans chk-2 gene Proteins 0.000 description 1
- 101100327819 Caenorhabditis elegans chl-1 gene Proteins 0.000 description 1
- 101100064707 Caenorhabditis elegans eef-2 gene Proteins 0.000 description 1
- 101100348341 Caenorhabditis elegans gas-1 gene Proteins 0.000 description 1
- 101100123850 Caenorhabditis elegans her-1 gene Proteins 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 229940127291 Calcium channel antagonist Drugs 0.000 description 1
- 102000004657 Calcium-Calmodulin-Dependent Protein Kinase Type 2 Human genes 0.000 description 1
- 108010003721 Calcium-Calmodulin-Dependent Protein Kinase Type 2 Proteins 0.000 description 1
- 101710155556 Calcium-dependent protease Proteins 0.000 description 1
- 102100033620 Calponin-1 Human genes 0.000 description 1
- 101710160269 Calreticulin-2 Proteins 0.000 description 1
- 102100038613 Calreticulin-3 Human genes 0.000 description 1
- 101710160266 Calreticulin-3 Proteins 0.000 description 1
- 101710187022 Cannabinoid receptor 2 Proteins 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 102100033377 Carbohydrate sulfotransferase 15 Human genes 0.000 description 1
- 108010027522 Carbonic Anhydrase III Proteins 0.000 description 1
- 102100033040 Carbonic anhydrase 12 Human genes 0.000 description 1
- 102100033041 Carbonic anhydrase 13 Human genes 0.000 description 1
- 101710094324 Carbonic anhydrase 13 Proteins 0.000 description 1
- 102100033007 Carbonic anhydrase 14 Human genes 0.000 description 1
- 102100024633 Carbonic anhydrase 2 Human genes 0.000 description 1
- 101710167917 Carbonic anhydrase 2 Proteins 0.000 description 1
- 102100024650 Carbonic anhydrase 3 Human genes 0.000 description 1
- 102100024644 Carbonic anhydrase 4 Human genes 0.000 description 1
- 101710167916 Carbonic anhydrase 4 Proteins 0.000 description 1
- 102100036372 Carbonic anhydrase 5A, mitochondrial Human genes 0.000 description 1
- 101710133954 Carbonic anhydrase 5A, mitochondrial Proteins 0.000 description 1
- 102100036376 Carbonic anhydrase 5B, mitochondrial Human genes 0.000 description 1
- 101710203682 Carbonic anhydrase 5B, mitochondrial Proteins 0.000 description 1
- 102100036369 Carbonic anhydrase 6 Human genes 0.000 description 1
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 1
- 108050007596 Carbonic anhydrase VII Proteins 0.000 description 1
- 102000028295 Carbonic anhydrase VII Human genes 0.000 description 1
- 102100036375 Carbonic anhydrase-related protein Human genes 0.000 description 1
- 101710086403 Carbonic anhydrase-related protein Proteins 0.000 description 1
- 102100032566 Carbonic anhydrase-related protein 10 Human genes 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- SHHKQEUPHAENFK-UHFFFAOYSA-N Carboquone Chemical compound O=C1C(C)=C(N2CC2)C(=O)C(C(COC(N)=O)OC)=C1N1CC1 SHHKQEUPHAENFK-UHFFFAOYSA-N 0.000 description 1
- 102100030613 Carboxypeptidase A1 Human genes 0.000 description 1
- 102100030621 Carboxypeptidase A4 Human genes 0.000 description 1
- 101710097783 Carboxypeptidase A4 Proteins 0.000 description 1
- 102100035024 Carboxypeptidase B Human genes 0.000 description 1
- 102100032378 Carboxypeptidase E Human genes 0.000 description 1
- 108010058255 Carboxypeptidase H Proteins 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 102100024533 Carcinoembryonic antigen-related cell adhesion molecule 1 Human genes 0.000 description 1
- 208000020446 Cardiac disease Diseases 0.000 description 1
- 102100028892 Cardiotrophin-1 Human genes 0.000 description 1
- AOCCBINRVIKJHY-UHFFFAOYSA-N Carmofur Chemical compound CCCCCCNC(=O)N1C=C(F)C(=O)NC1=O AOCCBINRVIKJHY-UHFFFAOYSA-N 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- 208000022621 Caronte Diseases 0.000 description 1
- 108700005376 Cartilage Oligomeric Matrix Proteins 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 108090000425 Caspase 6 Proteins 0.000 description 1
- 102000004018 Caspase 6 Human genes 0.000 description 1
- 108090000567 Caspase 7 Proteins 0.000 description 1
- 108090000426 Caspase-1 Proteins 0.000 description 1
- 102100035904 Caspase-1 Human genes 0.000 description 1
- 102100026549 Caspase-10 Human genes 0.000 description 1
- 108090000572 Caspase-10 Proteins 0.000 description 1
- 102000004066 Caspase-12 Human genes 0.000 description 1
- 108090000570 Caspase-12 Proteins 0.000 description 1
- 108090000552 Caspase-2 Proteins 0.000 description 1
- 102100032616 Caspase-2 Human genes 0.000 description 1
- 102100029855 Caspase-3 Human genes 0.000 description 1
- 101710090338 Caspase-4 Proteins 0.000 description 1
- 102100025597 Caspase-4 Human genes 0.000 description 1
- 102100038902 Caspase-7 Human genes 0.000 description 1
- 102100026548 Caspase-8 Human genes 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000712 Cathepsin B Proteins 0.000 description 1
- 102000004225 Cathepsin B Human genes 0.000 description 1
- 102000003902 Cathepsin C Human genes 0.000 description 1
- 108090000267 Cathepsin C Proteins 0.000 description 1
- 102000003908 Cathepsin D Human genes 0.000 description 1
- 108090000258 Cathepsin D Proteins 0.000 description 1
- 102000004178 Cathepsin E Human genes 0.000 description 1
- 108090000611 Cathepsin E Proteins 0.000 description 1
- 108090000610 Cathepsin F Proteins 0.000 description 1
- 102000004176 Cathepsin F Human genes 0.000 description 1
- 108090000619 Cathepsin H Proteins 0.000 description 1
- 102400001321 Cathepsin L Human genes 0.000 description 1
- 108090000624 Cathepsin L Proteins 0.000 description 1
- 102100026540 Cathepsin L2 Human genes 0.000 description 1
- 101710177066 Cathepsin O Proteins 0.000 description 1
- 108090000613 Cathepsin S Proteins 0.000 description 1
- 102100035654 Cathepsin S Human genes 0.000 description 1
- 102100026657 Cathepsin Z Human genes 0.000 description 1
- 108010061117 Cathepsin Z Proteins 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 102100031667 Cell adhesion molecule-related/down-regulated by oncogenes Human genes 0.000 description 1
- 101710114824 Cell death regulator Aven Proteins 0.000 description 1
- 101000716536 Centruroides sculpturatus Beta-toxin CsEI Proteins 0.000 description 1
- 108090000751 Ceramidases Proteins 0.000 description 1
- 241000202252 Cerberus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010036867 Cerebroside-Sulfatase Proteins 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 108010083702 Chemokine CCL21 Proteins 0.000 description 1
- 108010083647 Chemokine CCL24 Proteins 0.000 description 1
- 108010083698 Chemokine CCL26 Proteins 0.000 description 1
- 108010078239 Chemokine CX3CL1 Proteins 0.000 description 1
- 102000016950 Chemokine CXCL1 Human genes 0.000 description 1
- 108010014419 Chemokine CXCL1 Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 241000288673 Chiroptera Species 0.000 description 1
- 102100038196 Chitinase-3-like protein 1 Human genes 0.000 description 1
- JWBOIMRXGHLCPP-UHFFFAOYSA-N Chloditan Chemical compound C=1C=CC=C(Cl)C=1C(C(Cl)Cl)C1=CC=C(Cl)C=C1 JWBOIMRXGHLCPP-UHFFFAOYSA-N 0.000 description 1
- 102000011045 Chloride Channels Human genes 0.000 description 1
- 108010062745 Chloride Channels Proteins 0.000 description 1
- XCDXSSFOJZZGQC-UHFFFAOYSA-N Chlornaphazine Chemical compound C1=CC=CC2=CC(N(CCCl)CCCl)=CC=C21 XCDXSSFOJZZGQC-UHFFFAOYSA-N 0.000 description 1
- MKQWTWSXVILIKJ-LXGUWJNJSA-N Chlorozotocin Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)NC(=O)N(N=O)CCCl MKQWTWSXVILIKJ-LXGUWJNJSA-N 0.000 description 1
- 102100037327 Chondrolectin Human genes 0.000 description 1
- 101710084930 Chondrolectin Proteins 0.000 description 1
- 102100032765 Chordin-like protein 1 Human genes 0.000 description 1
- 102100032766 Chordin-like protein 2 Human genes 0.000 description 1
- 102100040484 Claspin Human genes 0.000 description 1
- 101710117926 Claspin Proteins 0.000 description 1
- 102100038449 Claudin-6 Human genes 0.000 description 1
- 108090000229 Claudin-6 Proteins 0.000 description 1
- 102000003780 Clusterin Human genes 0.000 description 1
- 108090000197 Clusterin Proteins 0.000 description 1
- 102100033577 Clusterin-like protein 1 Human genes 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 102100023804 Coagulation factor VII Human genes 0.000 description 1
- 102100029117 Coagulation factor X Human genes 0.000 description 1
- 102100030563 Coagulation factor XI Human genes 0.000 description 1
- 108010016640 Cobra Cardiotoxin Proteins Proteins 0.000 description 1
- 102100035932 Cocaine- and amphetamine-regulated transcript protein Human genes 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 description 1
- 101710194650 Collectin-12 Proteins 0.000 description 1
- 102100025890 Complement C1q tumor necrosis factor-related protein 3 Human genes 0.000 description 1
- 101710204075 Complement C1q tumor necrosis factor-related protein 3 Proteins 0.000 description 1
- 102100022133 Complement C3 Human genes 0.000 description 1
- 102100031506 Complement C5 Human genes 0.000 description 1
- 108090000044 Complement Factor I Proteins 0.000 description 1
- 102100025877 Complement component C1q receptor Human genes 0.000 description 1
- 102100025680 Complement decay-accelerating factor Human genes 0.000 description 1
- 108090000056 Complement factor B Proteins 0.000 description 1
- 102000003712 Complement factor B Human genes 0.000 description 1
- 102000003706 Complement factor D Human genes 0.000 description 1
- 108090000059 Complement factor D Proteins 0.000 description 1
- 102100035325 Complement factor H-related protein 5 Human genes 0.000 description 1
- 102100035431 Complement factor I Human genes 0.000 description 1
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 1
- 108010069241 Connexin 43 Proteins 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 108010087195 Contactin 1 Proteins 0.000 description 1
- 108010087196 Contactin 2 Proteins 0.000 description 1
- 102100024326 Contactin-1 Human genes 0.000 description 1
- 102100024340 Contactin-4 Human genes 0.000 description 1
- 101710107714 Contactin-4 Proteins 0.000 description 1
- 102100024343 Contactin-5 Human genes 0.000 description 1
- 101710107709 Contactin-5 Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102100032202 Cornulin Human genes 0.000 description 1
- 101710148705 Cornulin Proteins 0.000 description 1
- 102100032165 Corticotropin-releasing factor-binding protein Human genes 0.000 description 1
- 241001125840 Coryphaenidae Species 0.000 description 1
- 102100025278 Coxsackievirus and adenovirus receptor Human genes 0.000 description 1
- 102100029376 Cryptochrome-1 Human genes 0.000 description 1
- 235000004035 Cryptotaenia japonica Nutrition 0.000 description 1
- 102100031096 Cubilin Human genes 0.000 description 1
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 1
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 1
- 102100025191 Cyclin-A2 Human genes 0.000 description 1
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 1
- 108010072220 Cyclophilin A Proteins 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 108010061641 Cystatin A Proteins 0.000 description 1
- 108010061635 Cystatin B Proteins 0.000 description 1
- 108010061642 Cystatin C Proteins 0.000 description 1
- 102100031237 Cystatin-A Human genes 0.000 description 1
- 102100026891 Cystatin-B Human genes 0.000 description 1
- 102100026897 Cystatin-C Human genes 0.000 description 1
- 102100028182 Cystatin-D Human genes 0.000 description 1
- 102100028188 Cystatin-F Human genes 0.000 description 1
- 102100038381 Cystatin-M Human genes 0.000 description 1
- 108010039724 Cysteine dioxygenase Proteins 0.000 description 1
- 108010019961 Cysteine-Rich Protein 61 Proteins 0.000 description 1
- 102100032759 Cysteine-rich motor neuron 1 protein Human genes 0.000 description 1
- 102100027350 Cysteine-rich secretory protein 2 Human genes 0.000 description 1
- 101710126127 Cysteine-rich secretory protein 2 Proteins 0.000 description 1
- 102100027367 Cysteine-rich secretory protein 3 Human genes 0.000 description 1
- 101710126281 Cysteine-rich secretory protein 3 Proteins 0.000 description 1
- 108010079245 Cystic Fibrosis Transmembrane Conductance Regulator Proteins 0.000 description 1
- 102000012605 Cystic Fibrosis Transmembrane Conductance Regulator Human genes 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 108050008072 Cytochrome c oxidase subunit IV Proteins 0.000 description 1
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 1
- 102100038493 Cytokine receptor-like factor 1 Human genes 0.000 description 1
- 101710194728 Cytokine receptor-like factor 1 Proteins 0.000 description 1
- 102100027816 Cytotoxic and regulatory T-cell molecule Human genes 0.000 description 1
- 102100026233 DAN domain family member 5 Human genes 0.000 description 1
- 108010014790 DAX-1 Orphan Nuclear Receptor Proteins 0.000 description 1
- 108010037897 DC-specific ICAM-3 grabbing nonintegrin Proteins 0.000 description 1
- 108010060248 DNA Ligase ATP Proteins 0.000 description 1
- 108010001132 DNA Polymerase beta Proteins 0.000 description 1
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 description 1
- 102100031867 DNA excision repair protein ERCC-6 Human genes 0.000 description 1
- 102100033195 DNA ligase 4 Human genes 0.000 description 1
- 102100022302 DNA polymerase beta Human genes 0.000 description 1
- 102100024829 DNA polymerase delta catalytic subunit Human genes 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102100022204 DNA-dependent protein kinase catalytic subunit Human genes 0.000 description 1
- 101710157074 DNA-dependent protein kinase catalytic subunit Proteins 0.000 description 1
- 101001082109 Danio rerio Eukaryotic translation initiation factor 4E-1B Proteins 0.000 description 1
- 101100503636 Danio rerio fyna gene Proteins 0.000 description 1
- 101100208241 Danio rerio thbs3a gene Proteins 0.000 description 1
- 101100314281 Danio rerio trappc11 gene Proteins 0.000 description 1
- 102100035784 Decorin Human genes 0.000 description 1
- 108090000738 Decorin Proteins 0.000 description 1
- 101710178505 Defensin-1 Proteins 0.000 description 1
- 101710178510 Defensin-2 Proteins 0.000 description 1
- 102100026662 Delta and Notch-like epidermal growth factor-related receptor Human genes 0.000 description 1
- 102100036462 Delta-like protein 1 Human genes 0.000 description 1
- 102100033553 Delta-like protein 4 Human genes 0.000 description 1
- NNJPGOLRFBJNIW-UHFFFAOYSA-N Demecolcine Natural products C1=C(OC)C(=O)C=C2C(NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-UHFFFAOYSA-N 0.000 description 1
- 102000004099 Deoxyribonuclease (Pyrimidine Dimer) Human genes 0.000 description 1
- 108010036364 Deoxyribonuclease IV (Phage T4-Induced) Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 102100036912 Desmin Human genes 0.000 description 1
- 108010044052 Desmin Proteins 0.000 description 1
- 108010057987 Desmodus rotundus salivary plasminogen activator alpha 1 Proteins 0.000 description 1
- 108010045579 Desmoglein 1 Proteins 0.000 description 1
- 102000005707 Desmoglein 2 Human genes 0.000 description 1
- 108010045583 Desmoglein 2 Proteins 0.000 description 1
- 108010032035 Desmoglein 3 Proteins 0.000 description 1
- 102000007577 Desmoglein 3 Human genes 0.000 description 1
- 102100037124 Developmental pluripotency-associated 5 protein Human genes 0.000 description 1
- 102100037126 Developmental pluripotency-associated protein 4 Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 1
- 101710099518 Dickkopf-related protein 1 Proteins 0.000 description 1
- 102100030091 Dickkopf-related protein 2 Human genes 0.000 description 1
- 101710099523 Dickkopf-related protein 2 Proteins 0.000 description 1
- 102100037985 Dickkopf-related protein 3 Human genes 0.000 description 1
- 101710099550 Dickkopf-related protein 3 Proteins 0.000 description 1
- 102100037986 Dickkopf-related protein 4 Human genes 0.000 description 1
- 101710099554 Dickkopf-related protein 4 Proteins 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- 101001130157 Dioclea sclerocarpa Lectin alpha chain Proteins 0.000 description 1
- 102100036966 Dipeptidyl aminopeptidase-like protein 6 Human genes 0.000 description 1
- 102100029921 Dipeptidyl peptidase 1 Human genes 0.000 description 1
- 101710087078 Dipeptidyl peptidase 1 Proteins 0.000 description 1
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 1
- 101710087012 Dipeptidyl-peptidase 7 Proteins 0.000 description 1
- 108050007106 Dishevelled-1 Proteins 0.000 description 1
- 108050007103 Dishevelled-3 Proteins 0.000 description 1
- 108700021041 Disintegrin Proteins 0.000 description 1
- 102100039673 Disintegrin and metalloproteinase domain-containing protein 10 Human genes 0.000 description 1
- 102100031112 Disintegrin and metalloproteinase domain-containing protein 12 Human genes 0.000 description 1
- 102100031113 Disintegrin and metalloproteinase domain-containing protein 15 Human genes 0.000 description 1
- 102100031116 Disintegrin and metalloproteinase domain-containing protein 19 Human genes 0.000 description 1
- 102100025979 Disintegrin and metalloproteinase domain-containing protein 33 Human genes 0.000 description 1
- 102100024364 Disintegrin and metalloproteinase domain-containing protein 8 Human genes 0.000 description 1
- 102100024361 Disintegrin and metalloproteinase domain-containing protein 9 Human genes 0.000 description 1
- 230000010777 Disulfide Reduction Effects 0.000 description 1
- 108010090047 Dopamine and cAMP-Regulated Phosphoprotein 32 Proteins 0.000 description 1
- 102000012749 Dopamine and cAMP-Regulated Phosphoprotein 32 Human genes 0.000 description 1
- 102100037713 Down syndrome cell adhesion molecule Human genes 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 101100393884 Drosophila melanogaster Glut1 gene Proteins 0.000 description 1
- 101100230254 Drosophila melanogaster Glut3 gene Proteins 0.000 description 1
- 101100044298 Drosophila melanogaster fand gene Proteins 0.000 description 1
- 101100295776 Drosophila melanogaster onecut gene Proteins 0.000 description 1
- 102000043859 Dynamin Human genes 0.000 description 1
- 108700021058 Dynamin Proteins 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 1
- 102100023226 Early growth response protein 1 Human genes 0.000 description 1
- 101710194146 Ecotin Proteins 0.000 description 1
- 102100040897 Embryonic growth/differentiation factor 1 Human genes 0.000 description 1
- 108010044063 Endocrine-Gland-Derived Vascular Endothelial Growth Factor Proteins 0.000 description 1
- 108010036395 Endoglin Proteins 0.000 description 1
- 101710081048 Endonuclease III Proteins 0.000 description 1
- 102100037696 Endonuclease V Human genes 0.000 description 1
- 101710168245 Endoplasmic reticulum aminopeptidase 1 Proteins 0.000 description 1
- 101800001739 Endorepellin Proteins 0.000 description 1
- 108010079505 Endostatins Proteins 0.000 description 1
- 108010009900 Endothelial Protein C Receptor Proteins 0.000 description 1
- 102000009839 Endothelial Protein C Receptor Human genes 0.000 description 1
- 102100038591 Endothelial cell-selective adhesion molecule Human genes 0.000 description 1
- 102400000792 Endothelial monocyte-activating polypeptide 2 Human genes 0.000 description 1
- 102400000686 Endothelin-1 Human genes 0.000 description 1
- 101800004490 Endothelin-1 Proteins 0.000 description 1
- 102100029112 Endothelin-converting enzyme 1 Human genes 0.000 description 1
- 108030001679 Endothelin-converting enzyme 1 Proteins 0.000 description 1
- 102100029113 Endothelin-converting enzyme 2 Human genes 0.000 description 1
- 101710144982 Endothelin-converting enzyme 2 Proteins 0.000 description 1
- SAMRUMKYXPVKPA-VFKOLLTISA-N Enocitabine Chemical compound O=C1N=C(NC(=O)CCCCCCCCCCCCCCCCCCCCC)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 SAMRUMKYXPVKPA-VFKOLLTISA-N 0.000 description 1
- 101710201246 Eomesodermin Proteins 0.000 description 1
- 101710139422 Eotaxin Proteins 0.000 description 1
- 108010055196 EphA2 Receptor Proteins 0.000 description 1
- 108010055191 EphA3 Receptor Proteins 0.000 description 1
- 108010055182 EphA5 Receptor Proteins 0.000 description 1
- 108010055207 EphA6 Receptor Proteins 0.000 description 1
- 108010055153 EphA7 Receptor Proteins 0.000 description 1
- 108010055155 EphA8 Receptor Proteins 0.000 description 1
- 108010055334 EphB2 Receptor Proteins 0.000 description 1
- 102100030322 Ephrin type-A receptor 1 Human genes 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 102100030324 Ephrin type-A receptor 3 Human genes 0.000 description 1
- 102100021605 Ephrin type-A receptor 5 Human genes 0.000 description 1
- 102100021606 Ephrin type-A receptor 7 Human genes 0.000 description 1
- 102100021601 Ephrin type-A receptor 8 Human genes 0.000 description 1
- 102100030779 Ephrin type-B receptor 1 Human genes 0.000 description 1
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 1
- 102100031982 Ephrin type-B receptor 3 Human genes 0.000 description 1
- 102100031983 Ephrin type-B receptor 4 Human genes 0.000 description 1
- 102100031984 Ephrin type-B receptor 6 Human genes 0.000 description 1
- 102000020086 Ephrin-A1 Human genes 0.000 description 1
- 108010043945 Ephrin-A1 Proteins 0.000 description 1
- 108010043942 Ephrin-A2 Proteins 0.000 description 1
- 102100033919 Ephrin-A2 Human genes 0.000 description 1
- 108010043940 Ephrin-A3 Proteins 0.000 description 1
- 102100033940 Ephrin-A3 Human genes 0.000 description 1
- 108010043938 Ephrin-A4 Proteins 0.000 description 1
- 102100033942 Ephrin-A4 Human genes 0.000 description 1
- 108010043939 Ephrin-A5 Proteins 0.000 description 1
- 102100033941 Ephrin-A5 Human genes 0.000 description 1
- 102100033946 Ephrin-B1 Human genes 0.000 description 1
- 108010044099 Ephrin-B1 Proteins 0.000 description 1
- 102100023721 Ephrin-B2 Human genes 0.000 description 1
- 108010044090 Ephrin-B2 Proteins 0.000 description 1
- 102100023733 Ephrin-B3 Human genes 0.000 description 1
- 108010044085 Ephrin-B3 Proteins 0.000 description 1
- 102100030323 Epigen Human genes 0.000 description 1
- 108010016906 Epigen Proteins 0.000 description 1
- 102100025471 Epiphycan Human genes 0.000 description 1
- 101800000155 Epiregulin Proteins 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 102100036725 Epithelial discoidin domain-containing receptor 1 Human genes 0.000 description 1
- 101710131668 Epithelial discoidin domain-containing receptor 1 Proteins 0.000 description 1
- OBMLHUPNRURLOK-XGRAFVIBSA-N Epitiostanol Chemical compound C1[C@@H]2S[C@@H]2C[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 OBMLHUPNRURLOK-XGRAFVIBSA-N 0.000 description 1
- 101150099168 Erap1 gene Proteins 0.000 description 1
- 241000289659 Erinaceidae Species 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 102100031690 Erythroid transcription factor Human genes 0.000 description 1
- 101710100588 Erythroid transcription factor Proteins 0.000 description 1
- 241000283257 Eschrichtius robustus Species 0.000 description 1
- 229930189413 Esperamicin Natural products 0.000 description 1
- 102100038595 Estrogen receptor Human genes 0.000 description 1
- 102100031855 Estrogen-related receptor gamma Human genes 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 101710205516 Exostosin-like 2 Proteins 0.000 description 1
- 101710205521 Exostosin-like 3 Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102100026693 FAS-associated death domain protein Human genes 0.000 description 1
- 101150081880 FGF1 gene Proteins 0.000 description 1
- 101150018370 FRK gene Proteins 0.000 description 1
- 101150018272 FYN gene Proteins 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010023321 Factor VII Proteins 0.000 description 1
- 108010074864 Factor XI Proteins 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 102100030421 Fatty acid-binding protein 5 Human genes 0.000 description 1
- 102100037735 Fatty acid-binding protein 9 Human genes 0.000 description 1
- 102100030431 Fatty acid-binding protein, adipocyte Human genes 0.000 description 1
- 102100037733 Fatty acid-binding protein, brain Human genes 0.000 description 1
- 102100037738 Fatty acid-binding protein, heart Human genes 0.000 description 1
- 102100026748 Fatty acid-binding protein, intestinal Human genes 0.000 description 1
- 102100026745 Fatty acid-binding protein, liver Human genes 0.000 description 1
- 108010021468 Fc gamma receptor IIA Proteins 0.000 description 1
- 108010021472 Fc gamma receptor IIB Proteins 0.000 description 1
- 108010021470 Fc gamma receptor IIC Proteins 0.000 description 1
- 101710120224 Fc receptor-like protein 1 Proteins 0.000 description 1
- 102100031512 Fc receptor-like protein 3 Human genes 0.000 description 1
- 241000701915 Feline panleukopenia virus Species 0.000 description 1
- 101100058548 Felis catus BMI1 gene Proteins 0.000 description 1
- 102100028044 Fetuin-B Human genes 0.000 description 1
- 108010086121 Fetuin-B Proteins 0.000 description 1
- 101150040897 Fgr gene Proteins 0.000 description 1
- 108090000569 Fibroblast Growth Factor-23 Proteins 0.000 description 1
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 description 1
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 description 1
- 108050003237 Fibroblast growth factor 11 Proteins 0.000 description 1
- 102100028413 Fibroblast growth factor 11 Human genes 0.000 description 1
- 108050003239 Fibroblast growth factor 12 Proteins 0.000 description 1
- 102100028417 Fibroblast growth factor 12 Human genes 0.000 description 1
- 108050002072 Fibroblast growth factor 16 Proteins 0.000 description 1
- 102100035307 Fibroblast growth factor 16 Human genes 0.000 description 1
- 108050002074 Fibroblast growth factor 17 Proteins 0.000 description 1
- 102100031734 Fibroblast growth factor 19 Human genes 0.000 description 1
- 108050002085 Fibroblast growth factor 20 Proteins 0.000 description 1
- 102100031361 Fibroblast growth factor 20 Human genes 0.000 description 1
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 description 1
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 description 1
- 102100024804 Fibroblast growth factor 22 Human genes 0.000 description 1
- 108050002062 Fibroblast growth factor 22 Proteins 0.000 description 1
- 102100024802 Fibroblast growth factor 23 Human genes 0.000 description 1
- 108090000378 Fibroblast growth factor 3 Proteins 0.000 description 1
- 102100028043 Fibroblast growth factor 3 Human genes 0.000 description 1
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 1
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 description 1
- 108090000380 Fibroblast growth factor 5 Proteins 0.000 description 1
- 102100028073 Fibroblast growth factor 5 Human genes 0.000 description 1
- 108090000382 Fibroblast growth factor 6 Proteins 0.000 description 1
- 102100028075 Fibroblast growth factor 6 Human genes 0.000 description 1
- 108090000368 Fibroblast growth factor 8 Proteins 0.000 description 1
- 102100037680 Fibroblast growth factor 8 Human genes 0.000 description 1
- 102100037665 Fibroblast growth factor 9 Human genes 0.000 description 1
- 108090000367 Fibroblast growth factor 9 Proteins 0.000 description 1
- 102100023590 Fibroblast growth factor-binding protein 1 Human genes 0.000 description 1
- 101710094971 Fibroblast growth factor-binding protein 1 Proteins 0.000 description 1
- 102100028065 Fibulin-5 Human genes 0.000 description 1
- 101710170766 Fibulin-5 Proteins 0.000 description 1
- 102100024521 Ficolin-2 Human genes 0.000 description 1
- 101710155249 Ficolin-2 Proteins 0.000 description 1
- 102100024520 Ficolin-3 Human genes 0.000 description 1
- 101710155250 Ficolin-3 Proteins 0.000 description 1
- 108090000652 Flap endonucleases Proteins 0.000 description 1
- 102000004150 Flap endonucleases Human genes 0.000 description 1
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 1
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 108010014612 Follistatin Proteins 0.000 description 1
- 102000016970 Follistatin Human genes 0.000 description 1
- 108010012820 Follistatin-Related Proteins Proteins 0.000 description 1
- 102100029378 Follistatin-related protein 1 Human genes 0.000 description 1
- 102100029379 Follistatin-related protein 3 Human genes 0.000 description 1
- 102100021084 Forkhead box protein C1 Human genes 0.000 description 1
- 102100037057 Forkhead box protein D1 Human genes 0.000 description 1
- 102100037060 Forkhead box protein D3 Human genes 0.000 description 1
- 101710181403 Frizzled Proteins 0.000 description 1
- 102100021259 Frizzled-1 Human genes 0.000 description 1
- 101710140948 Frizzled-1 Proteins 0.000 description 1
- 102100021265 Frizzled-2 Human genes 0.000 description 1
- 101710140946 Frizzled-2 Proteins 0.000 description 1
- 102100021262 Frizzled-3 Human genes 0.000 description 1
- 101710140944 Frizzled-3 Proteins 0.000 description 1
- 102100039820 Frizzled-4 Human genes 0.000 description 1
- 108050007986 Frizzled-4 Proteins 0.000 description 1
- 102100039818 Frizzled-5 Human genes 0.000 description 1
- 101710140951 Frizzled-5 Proteins 0.000 description 1
- 102100039799 Frizzled-6 Human genes 0.000 description 1
- 108050008000 Frizzled-6 Proteins 0.000 description 1
- 102100039676 Frizzled-7 Human genes 0.000 description 1
- 108050007985 Frizzled-7 Proteins 0.000 description 1
- 102100028461 Frizzled-9 Human genes 0.000 description 1
- 101710140940 Frizzled-9 Proteins 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 102100023416 G-protein coupled receptor 15 Human genes 0.000 description 1
- 102100030280 G-protein coupled receptor 39 Human genes 0.000 description 1
- 101710142641 G-protein coupled receptor-associated sorting protein 1 Proteins 0.000 description 1
- 101710142639 G-protein coupled receptor-associated sorting protein 2 Proteins 0.000 description 1
- 101150022345 GAS6 gene Proteins 0.000 description 1
- 102100033324 GATA zinc finger domain-containing protein 1 Human genes 0.000 description 1
- 101710082961 GATA-binding factor 2 Proteins 0.000 description 1
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 102000008412 GATA5 Transcription Factor Human genes 0.000 description 1
- 108010021779 GATA5 Transcription Factor Proteins 0.000 description 1
- 101150014889 Gad1 gene Proteins 0.000 description 1
- 108010001498 Galectin 1 Proteins 0.000 description 1
- 108010001496 Galectin 2 Proteins 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102000000805 Galectin 4 Human genes 0.000 description 1
- 102100021736 Galectin-1 Human genes 0.000 description 1
- 102100021735 Galectin-2 Human genes 0.000 description 1
- 102000044465 Galectin-7 Human genes 0.000 description 1
- 102000044445 Galectin-8 Human genes 0.000 description 1
- 101710121810 Galectin-9 Proteins 0.000 description 1
- 102100031351 Galectin-9 Human genes 0.000 description 1
- 101001066288 Gallus gallus GATA-binding factor 3 Proteins 0.000 description 1
- 102100035924 Gamma-aminobutyric acid type B receptor subunit 2 Human genes 0.000 description 1
- 101710086265 Gamma-aminobutyric acid type B receptor subunit 2 Proteins 0.000 description 1
- 101710115997 Gamma-tubulin complex component 2 Proteins 0.000 description 1
- 102100021337 Gap junction alpha-1 protein Human genes 0.000 description 1
- 102400000921 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 108700004714 Gelonium multiflorum GEL Proteins 0.000 description 1
- 102100031885 General transcription and DNA repair factor IIH helicase subunit XPB Human genes 0.000 description 1
- 108090000495 Glia Maturation Factor Proteins 0.000 description 1
- 102000004038 Glia Maturation Factor Human genes 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](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(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 1
- 102100033417 Glucocorticoid receptor Human genes 0.000 description 1
- 108010017544 Glucosylceramidase Proteins 0.000 description 1
- 102000004547 Glucosylceramidase Human genes 0.000 description 1
- 108010058940 Glutamyl Aminopeptidase Proteins 0.000 description 1
- 102100025783 Glutamyl aminopeptidase Human genes 0.000 description 1
- 102000017278 Glutaredoxin Human genes 0.000 description 1
- 108050005205 Glutaredoxin Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102000007390 Glycogen Phosphorylase Human genes 0.000 description 1
- 108010046163 Glycogen Phosphorylase Proteins 0.000 description 1
- 102100035716 Glycophorin-A Human genes 0.000 description 1
- 108091005250 Glycophorins Proteins 0.000 description 1
- 108050009388 Glypican-2 Proteins 0.000 description 1
- 108050007237 Glypican-3 Proteins 0.000 description 1
- 108050009377 Glypican-5 Proteins 0.000 description 1
- 108050009385 Glypican-6 Proteins 0.000 description 1
- 102000001989 Glypican-6 Human genes 0.000 description 1
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 description 1
- 108010069236 Goserelin Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 101710108699 Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Proteins 0.000 description 1
- 102100028113 Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Human genes 0.000 description 1
- 102100021186 Granulysin Human genes 0.000 description 1
- 101710168479 Granulysin Proteins 0.000 description 1
- 101710113248 Granzyme D Proteins 0.000 description 1
- 101710113211 Granzyme G Proteins 0.000 description 1
- 102100038393 Granzyme H Human genes 0.000 description 1
- 101710113220 Granzyme H Proteins 0.000 description 1
- 102100038367 Gremlin-1 Human genes 0.000 description 1
- 108010090296 Growth Differentiation Factor 1 Proteins 0.000 description 1
- 108010090290 Growth Differentiation Factor 2 Proteins 0.000 description 1
- 108010090293 Growth Differentiation Factor 3 Proteins 0.000 description 1
- 108010090254 Growth Differentiation Factor 5 Proteins 0.000 description 1
- 108010090250 Growth Differentiation Factor 6 Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 239000000095 Growth Hormone-Releasing Hormone Substances 0.000 description 1
- 102100033067 Growth factor receptor-bound protein 2 Human genes 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 101710099093 Growth hormone receptor Proteins 0.000 description 1
- 102100040892 Growth/differentiation factor 2 Human genes 0.000 description 1
- 102100035364 Growth/differentiation factor 3 Human genes 0.000 description 1
- 102100035379 Growth/differentiation factor 5 Human genes 0.000 description 1
- 102100035368 Growth/differentiation factor 6 Human genes 0.000 description 1
- 102100035363 Growth/differentiation factor 7 Human genes 0.000 description 1
- 101710204283 Growth/differentiation factor 7 Proteins 0.000 description 1
- 102100039939 Growth/differentiation factor 8 Human genes 0.000 description 1
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- 108010066705 H-cadherin Proteins 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 102100030595 HLA class II histocompatibility antigen gamma chain Human genes 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 description 1
- 101000588935 Heteractis crispa Delta-stichotoxin-Hcr1b Proteins 0.000 description 1
- 101000588929 Heteractis crispa Delta-stichotoxin-Hcr1e Proteins 0.000 description 1
- 101000588275 Heteractis magnifica Delta-stichotoxin-Rpa1b Proteins 0.000 description 1
- 102100029217 High affinity cationic amino acid transporter 1 Human genes 0.000 description 1
- 102000007625 Hirudins Human genes 0.000 description 1
- 108010007267 Hirudins Proteins 0.000 description 1
- 102000030789 Histidine Ammonia-Lyase Human genes 0.000 description 1
- 108700006308 Histidine ammonia-lyases Proteins 0.000 description 1
- 101710141457 Histidine-binding periplasmic protein Proteins 0.000 description 1
- 108091016366 Histone-lysine N-methyltransferase EHMT1 Proteins 0.000 description 1
- 102100035042 Histone-lysine N-methyltransferase EHMT2 Human genes 0.000 description 1
- 102100031670 Homeobox protein CDX-4 Human genes 0.000 description 1
- 101710160857 Homeobox protein Hox-B3 Proteins 0.000 description 1
- 101710123483 Homeobox protein Hox-D11 Proteins 0.000 description 1
- 101000964898 Homo sapiens 14-3-3 protein zeta/delta Proteins 0.000 description 1
- 101000742701 Homo sapiens 5'-AMP-activated protein kinase subunit beta-1 Proteins 0.000 description 1
- 101000742799 Homo sapiens 5'-AMP-activated protein kinase subunit beta-2 Proteins 0.000 description 1
- 101000727998 Homo sapiens ADAMTS-like protein 1 Proteins 0.000 description 1
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 1
- 101000799140 Homo sapiens Activin receptor type-1 Proteins 0.000 description 1
- 101000718243 Homo sapiens Adhesion G protein-coupled receptor E5 Proteins 0.000 description 1
- 101000720039 Homo sapiens Aminoacylase-1 Proteins 0.000 description 1
- 101000776165 Homo sapiens Amphoterin-induced protein 2 Proteins 0.000 description 1
- 101000776186 Homo sapiens Amphoterin-induced protein 3 Proteins 0.000 description 1
- 101000693093 Homo sapiens Angiopoietin-related protein 1 Proteins 0.000 description 1
- 101000693081 Homo sapiens Angiopoietin-related protein 2 Proteins 0.000 description 1
- 101000693085 Homo sapiens Angiopoietin-related protein 3 Proteins 0.000 description 1
- 101000693076 Homo sapiens Angiopoietin-related protein 4 Proteins 0.000 description 1
- 101000796085 Homo sapiens Anthrax toxin receptor 2 Proteins 0.000 description 1
- 101000793362 Homo sapiens Apelin receptor Proteins 0.000 description 1
- 101000890622 Homo sapiens Apoptosis-inducing factor 1, mitochondrial Proteins 0.000 description 1
- 101000901140 Homo sapiens Arylsulfatase A Proteins 0.000 description 1
- 101000785944 Homo sapiens Asialoglycoprotein receptor 1 Proteins 0.000 description 1
- 101000874566 Homo sapiens Axin-1 Proteins 0.000 description 1
- 101000793686 Homo sapiens Azurocidin Proteins 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000934359 Homo sapiens B-cell differentiation antigen CD72 Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000740545 Homo sapiens BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Proteins 0.000 description 1
- 101000934632 Homo sapiens BMP-binding endothelial regulator protein Proteins 0.000 description 1
- 101000896234 Homo sapiens Baculoviral IAP repeat-containing protein 5 Proteins 0.000 description 1
- 101000798441 Homo sapiens Basigin Proteins 0.000 description 1
- 101000899346 Homo sapiens Bcl-2-related ovarian killer protein Proteins 0.000 description 1
- 101000766145 Homo sapiens Beta-1,4-galactosyltransferase 1 Proteins 0.000 description 1
- 101000685668 Homo sapiens Bile acyl-CoA synthetase Proteins 0.000 description 1
- 101000899368 Homo sapiens Bone morphogenetic protein 8B Proteins 0.000 description 1
- 101000777558 Homo sapiens C-C chemokine receptor type 10 Proteins 0.000 description 1
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 description 1
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 1
- 101000716063 Homo sapiens C-C chemokine receptor type 8 Proteins 0.000 description 1
- 101000716070 Homo sapiens C-C chemokine receptor type 9 Proteins 0.000 description 1
- 101000978379 Homo sapiens C-C motif chemokine 13 Proteins 0.000 description 1
- 101000978381 Homo sapiens C-C motif chemokine 14 Proteins 0.000 description 1
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 description 1
- 101000978375 Homo sapiens C-C motif chemokine 16 Proteins 0.000 description 1
- 101000978371 Homo sapiens C-C motif chemokine 18 Proteins 0.000 description 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 1
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 1
- 101000713078 Homo sapiens C-C motif chemokine 24 Proteins 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 description 1
- 101000897477 Homo sapiens C-C motif chemokine 28 Proteins 0.000 description 1
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 1
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 description 1
- 101000916050 Homo sapiens C-X-C chemokine receptor type 3 Proteins 0.000 description 1
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 1
- 101000922405 Homo sapiens C-X-C chemokine receptor type 5 Proteins 0.000 description 1
- 101000856683 Homo sapiens C-X-C chemokine receptor type 6 Proteins 0.000 description 1
- 101000858088 Homo sapiens C-X-C motif chemokine 10 Proteins 0.000 description 1
- 101000858060 Homo sapiens C-X-C motif chemokine 11 Proteins 0.000 description 1
- 101000889128 Homo sapiens C-X-C motif chemokine 2 Proteins 0.000 description 1
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 description 1
- 101000947177 Homo sapiens C-X-C motif chemokine 6 Proteins 0.000 description 1
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 description 1
- 101000942296 Homo sapiens C-type lectin domain family 10 member A Proteins 0.000 description 1
- 101000766907 Homo sapiens C-type lectin domain family 4 member C Proteins 0.000 description 1
- 101000766905 Homo sapiens C-type lectin domain family 4 member D Proteins 0.000 description 1
- 101000749322 Homo sapiens C-type lectin domain family 6 member A Proteins 0.000 description 1
- 101000749325 Homo sapiens C-type lectin domain family 7 member A Proteins 0.000 description 1
- 101000914211 Homo sapiens CASP8 and FADD-like apoptosis regulator Proteins 0.000 description 1
- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 description 1
- 101000919678 Homo sapiens CCR4-NOT transcription complex subunit 11 Proteins 0.000 description 1
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 description 1
- 101000884298 Homo sapiens CD226 antigen Proteins 0.000 description 1
- 101000934335 Homo sapiens CD320 antigen Proteins 0.000 description 1
- 101000716130 Homo sapiens CD48 antigen Proteins 0.000 description 1
- 101000911996 Homo sapiens CD5 antigen-like Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 101000860854 Homo sapiens COUP transcription factor 1 Proteins 0.000 description 1
- 101000860860 Homo sapiens COUP transcription factor 2 Proteins 0.000 description 1
- 101000737742 Homo sapiens CUB domain-containing protein 1 Proteins 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000875075 Homo sapiens Cannabinoid receptor 2 Proteins 0.000 description 1
- 101000867836 Homo sapiens Carbonic anhydrase-related protein 10 Proteins 0.000 description 1
- 101000772551 Homo sapiens Carboxypeptidase A1 Proteins 0.000 description 1
- 101000946524 Homo sapiens Carboxypeptidase B Proteins 0.000 description 1
- 101000981093 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 1 Proteins 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 1
- 101000761509 Homo sapiens Cathepsin K Proteins 0.000 description 1
- 101000983577 Homo sapiens Cathepsin L2 Proteins 0.000 description 1
- 101000910979 Homo sapiens Cathepsin Z Proteins 0.000 description 1
- 101000698131 Homo sapiens Cell death regulator Aven Proteins 0.000 description 1
- 101000851684 Homo sapiens Chimeric ERCC6-PGBD3 protein Proteins 0.000 description 1
- 101000883515 Homo sapiens Chitinase-3-like protein 1 Proteins 0.000 description 1
- 101000879661 Homo sapiens Chitotriosidase-1 Proteins 0.000 description 1
- 101000941971 Homo sapiens Chordin-like protein 1 Proteins 0.000 description 1
- 101000941976 Homo sapiens Chordin-like protein 2 Proteins 0.000 description 1
- 101000945106 Homo sapiens Clusterin-like protein 1 Proteins 0.000 description 1
- 101000715592 Homo sapiens Cocaine- and amphetamine-regulated transcript protein Proteins 0.000 description 1
- 101000909528 Homo sapiens Collectin-12 Proteins 0.000 description 1
- 101000901154 Homo sapiens Complement C3 Proteins 0.000 description 1
- 101000941598 Homo sapiens Complement C5 Proteins 0.000 description 1
- 101000933665 Homo sapiens Complement component C1q receptor Proteins 0.000 description 1
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 description 1
- 101000878134 Homo sapiens Complement factor H-related protein 5 Proteins 0.000 description 1
- 101000921095 Homo sapiens Corticotropin-releasing factor-binding protein Proteins 0.000 description 1
- 101000858031 Homo sapiens Coxsackievirus and adenovirus receptor Proteins 0.000 description 1
- 101001047110 Homo sapiens Creatine kinase M-type Proteins 0.000 description 1
- 101000919351 Homo sapiens Cryptochrome-1 Proteins 0.000 description 1
- 101000868333 Homo sapiens Cyclin-dependent kinase 1 Proteins 0.000 description 1
- 101000916687 Homo sapiens Cystatin-D Proteins 0.000 description 1
- 101000916688 Homo sapiens Cystatin-F Proteins 0.000 description 1
- 101000884770 Homo sapiens Cystatin-M Proteins 0.000 description 1
- 101000942095 Homo sapiens Cysteine-rich motor neuron 1 protein Proteins 0.000 description 1
- 101000912351 Homo sapiens DAN domain family member 5 Proteins 0.000 description 1
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 description 1
- 101000920783 Homo sapiens DNA excision repair protein ERCC-6 Proteins 0.000 description 1
- 101000909198 Homo sapiens DNA polymerase delta catalytic subunit Proteins 0.000 description 1
- 101001054266 Homo sapiens Delta and Notch-like epidermal growth factor-related receptor Proteins 0.000 description 1
- 101000928537 Homo sapiens Delta-like protein 1 Proteins 0.000 description 1
- 101000872077 Homo sapiens Delta-like protein 4 Proteins 0.000 description 1
- 101000881848 Homo sapiens Developmental pluripotency-associated 5 protein Proteins 0.000 description 1
- 101000881868 Homo sapiens Developmental pluripotency-associated protein 4 Proteins 0.000 description 1
- 101000804935 Homo sapiens Dipeptidyl aminopeptidase-like protein 6 Proteins 0.000 description 1
- 101000931864 Homo sapiens Dipeptidyl peptidase 2 Proteins 0.000 description 1
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 1
- 101000777455 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 15 Proteins 0.000 description 1
- 101000777461 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 17 Proteins 0.000 description 1
- 101000777464 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 19 Proteins 0.000 description 1
- 101000720049 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 33 Proteins 0.000 description 1
- 101000832767 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 8 Proteins 0.000 description 1
- 101000832769 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 9 Proteins 0.000 description 1
- 101000880945 Homo sapiens Down syndrome cell adhesion molecule Proteins 0.000 description 1
- 101000877379 Homo sapiens ETS-related transcription factor Elf-3 Proteins 0.000 description 1
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 1
- 101001049697 Homo sapiens Early growth response protein 1 Proteins 0.000 description 1
- 101000898718 Homo sapiens Endoplasmic reticulum aminopeptidase 2 Proteins 0.000 description 1
- 101000882622 Homo sapiens Endothelial cell-selective adhesion molecule Proteins 0.000 description 1
- 101001064167 Homo sapiens Eomesodermin homolog Proteins 0.000 description 1
- 101000978392 Homo sapiens Eotaxin Proteins 0.000 description 1
- 101000938354 Homo sapiens Ephrin type-A receptor 1 Proteins 0.000 description 1
- 101001064150 Homo sapiens Ephrin type-B receptor 1 Proteins 0.000 description 1
- 101001064458 Homo sapiens Ephrin type-B receptor 3 Proteins 0.000 description 1
- 101001064451 Homo sapiens Ephrin type-B receptor 6 Proteins 0.000 description 1
- 101001056751 Homo sapiens Epiphycan Proteins 0.000 description 1
- 101000882584 Homo sapiens Estrogen receptor Proteins 0.000 description 1
- 101000875558 Homo sapiens Exostosin-like 2 Proteins 0.000 description 1
- 101000875556 Homo sapiens Exostosin-like 3 Proteins 0.000 description 1
- 101000911074 Homo sapiens FAS-associated death domain protein Proteins 0.000 description 1
- 101001062855 Homo sapiens Fatty acid-binding protein 5 Proteins 0.000 description 1
- 101001027665 Homo sapiens Fatty acid-binding protein 9 Proteins 0.000 description 1
- 101001062864 Homo sapiens Fatty acid-binding protein, adipocyte Proteins 0.000 description 1
- 101001027674 Homo sapiens Fatty acid-binding protein, brain Proteins 0.000 description 1
- 101001027663 Homo sapiens Fatty acid-binding protein, heart Proteins 0.000 description 1
- 101000911337 Homo sapiens Fatty acid-binding protein, intestinal Proteins 0.000 description 1
- 101000911317 Homo sapiens Fatty acid-binding protein, liver Proteins 0.000 description 1
- 101000846913 Homo sapiens Fc receptor-like protein 1 Proteins 0.000 description 1
- 101000846910 Homo sapiens Fc receptor-like protein 3 Proteins 0.000 description 1
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 1
- 101001062535 Homo sapiens Follistatin-related protein 1 Proteins 0.000 description 1
- 101000818310 Homo sapiens Forkhead box protein C1 Proteins 0.000 description 1
- 101001029317 Homo sapiens Forkhead box protein D1 Proteins 0.000 description 1
- 101001029308 Homo sapiens Forkhead box protein D3 Proteins 0.000 description 1
- 101000854520 Homo sapiens Fractalkine Proteins 0.000 description 1
- 101001061408 Homo sapiens Frizzled-8 Proteins 0.000 description 1
- 101000829794 Homo sapiens G-protein coupled receptor 15 Proteins 0.000 description 1
- 101001009541 Homo sapiens G-protein coupled receptor 39 Proteins 0.000 description 1
- 101000926786 Homo sapiens GATA zinc finger domain-containing protein 1 Proteins 0.000 description 1
- 101000608772 Homo sapiens Galectin-7 Proteins 0.000 description 1
- 101000608769 Homo sapiens Galectin-8 Proteins 0.000 description 1
- 101000920748 Homo sapiens General transcription and DNA repair factor IIH helicase subunit XPB Proteins 0.000 description 1
- 101000926939 Homo sapiens Glucocorticoid receptor Proteins 0.000 description 1
- 101000873546 Homo sapiens Glutamate decarboxylase 1 Proteins 0.000 description 1
- 101000719019 Homo sapiens Glutamyl aminopeptidase Proteins 0.000 description 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001032872 Homo sapiens Gremlin-1 Proteins 0.000 description 1
- 101000871017 Homo sapiens Growth factor receptor-bound protein 2 Proteins 0.000 description 1
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 description 1
- 101001082627 Homo sapiens HLA class II histocompatibility antigen gamma chain Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000972946 Homo sapiens Hepatocyte growth factor receptor Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101000877314 Homo sapiens Histone-lysine N-methyltransferase EHMT1 Proteins 0.000 description 1
- 101000877312 Homo sapiens Histone-lysine N-methyltransferase EHMT2 Proteins 0.000 description 1
- 101000777790 Homo sapiens Homeobox protein CDX-4 Proteins 0.000 description 1
- 101000839775 Homo sapiens Homeobox protein Hox-B3 Proteins 0.000 description 1
- 101000962591 Homo sapiens Homeobox protein Hox-D11 Proteins 0.000 description 1
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 1
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 1
- 101001050318 Homo sapiens Junctional adhesion molecule-like Proteins 0.000 description 1
- 101001027146 Homo sapiens Kelch domain-containing protein 10 Proteins 0.000 description 1
- 101001046960 Homo sapiens Keratin, type II cytoskeletal 1 Proteins 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 101001059438 Homo sapiens Leucine-rich repeat transmembrane protein FLRT1 Proteins 0.000 description 1
- 101000893526 Homo sapiens Leucine-rich repeat transmembrane protein FLRT2 Proteins 0.000 description 1
- 101000893530 Homo sapiens Leucine-rich repeat transmembrane protein FLRT3 Proteins 0.000 description 1
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 description 1
- 101000608935 Homo sapiens Leukosialin Proteins 0.000 description 1
- 101000685655 Homo sapiens Long-chain fatty acid transport protein 1 Proteins 0.000 description 1
- 101000685660 Homo sapiens Long-chain fatty acid transport protein 4 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101000624625 Homo sapiens M-phase inducer phosphatase 1 Proteins 0.000 description 1
- 101000624631 Homo sapiens M-phase inducer phosphatase 2 Proteins 0.000 description 1
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 1
- 101000961414 Homo sapiens Membrane cofactor protein Proteins 0.000 description 1
- 101000950710 Homo sapiens Mitogen-activated protein kinase 6 Proteins 0.000 description 1
- 101001018196 Homo sapiens Mitogen-activated protein kinase kinase kinase 5 Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101000818546 Homo sapiens N-formyl peptide receptor 2 Proteins 0.000 description 1
- 101001059802 Homo sapiens N-formyl peptide receptor 3 Proteins 0.000 description 1
- 101000971513 Homo sapiens Natural killer cells antigen CD94 Proteins 0.000 description 1
- 101000602237 Homo sapiens Neuroblastoma suppressor of tumorigenicity 1 Proteins 0.000 description 1
- 101000979249 Homo sapiens Neuromodulin Proteins 0.000 description 1
- 101000975757 Homo sapiens Neutral ceramidase Proteins 0.000 description 1
- 101000603882 Homo sapiens Nuclear receptor subfamily 1 group I member 3 Proteins 0.000 description 1
- 101001109682 Homo sapiens Nuclear receptor subfamily 6 group A member 1 Proteins 0.000 description 1
- 101000992164 Homo sapiens One cut domain family member 2 Proteins 0.000 description 1
- 101000878221 Homo sapiens Peptidyl-prolyl cis-trans isomerase FKBP8 Proteins 0.000 description 1
- 101000586618 Homo sapiens Poliovirus receptor Proteins 0.000 description 1
- 101001113490 Homo sapiens Poly(A)-specific ribonuclease PARN Proteins 0.000 description 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 1
- 101000684208 Homo sapiens Prolyl endopeptidase FAP Proteins 0.000 description 1
- 101000859935 Homo sapiens Protein CREG1 Proteins 0.000 description 1
- 101000891860 Homo sapiens Protein FAM3A Proteins 0.000 description 1
- 101000891842 Homo sapiens Protein FAM3B Proteins 0.000 description 1
- 101000891845 Homo sapiens Protein FAM3C Proteins 0.000 description 1
- 101000891848 Homo sapiens Protein FAM3D Proteins 0.000 description 1
- 101000911388 Homo sapiens Protein FAM98A Proteins 0.000 description 1
- 101000931462 Homo sapiens Protein FosB Proteins 0.000 description 1
- 101000836765 Homo sapiens Protein amnionless Proteins 0.000 description 1
- 101000930354 Homo sapiens Protein dispatched homolog 1 Proteins 0.000 description 1
- 101000919288 Homo sapiens Protein disulfide isomerase CRELD1 Proteins 0.000 description 1
- 101000919297 Homo sapiens Protein disulfide isomerase CRELD2 Proteins 0.000 description 1
- 101001116931 Homo sapiens Protocadherin alpha-6 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101000643391 Homo sapiens Serine/arginine-rich splicing factor 11 Proteins 0.000 description 1
- 101000838578 Homo sapiens Serine/threonine-protein kinase TAO2 Proteins 0.000 description 1
- 101000651549 Homo sapiens Signal recognition particle subunit SRP68 Proteins 0.000 description 1
- 101000665150 Homo sapiens Small nuclear ribonucleoprotein Sm D1 Proteins 0.000 description 1
- 101000693265 Homo sapiens Sphingosine 1-phosphate receptor 1 Proteins 0.000 description 1
- 101000653759 Homo sapiens Sphingosine 1-phosphate receptor 5 Proteins 0.000 description 1
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 1
- 101000934376 Homo sapiens T-cell differentiation antigen CD6 Proteins 0.000 description 1
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 description 1
- 101100537522 Homo sapiens TNFSF13B gene Proteins 0.000 description 1
- 101000578693 Homo sapiens Target of rapamycin complex subunit LST8 Proteins 0.000 description 1
- 101000659879 Homo sapiens Thrombospondin-1 Proteins 0.000 description 1
- 101000633605 Homo sapiens Thrombospondin-2 Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 101000652332 Homo sapiens Transcription factor SOX-1 Proteins 0.000 description 1
- 101001137337 Homo sapiens Transcriptional activator protein Pur-alpha Proteins 0.000 description 1
- 101000685107 Homo sapiens Transcriptional repressor scratch 2 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 101000649014 Homo sapiens Triple functional domain protein Proteins 0.000 description 1
- 101000830565 Homo sapiens Tumor necrosis factor ligand superfamily member 10 Proteins 0.000 description 1
- 101000830603 Homo sapiens Tumor necrosis factor ligand superfamily member 11 Proteins 0.000 description 1
- 101000830598 Homo sapiens Tumor necrosis factor ligand superfamily member 12 Proteins 0.000 description 1
- 101000830596 Homo sapiens Tumor necrosis factor ligand superfamily member 15 Proteins 0.000 description 1
- 101000764263 Homo sapiens Tumor necrosis factor ligand superfamily member 4 Proteins 0.000 description 1
- 101000610609 Homo sapiens Tumor necrosis factor receptor superfamily member 10D Proteins 0.000 description 1
- 101000798130 Homo sapiens Tumor necrosis factor receptor superfamily member 11B Proteins 0.000 description 1
- 101000648507 Homo sapiens Tumor necrosis factor receptor superfamily member 14 Proteins 0.000 description 1
- 101000801254 Homo sapiens Tumor necrosis factor receptor superfamily member 16 Proteins 0.000 description 1
- 101000762805 Homo sapiens Tumor necrosis factor receptor superfamily member 19L Proteins 0.000 description 1
- 101000801232 Homo sapiens Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 1
- 101000679907 Homo sapiens Tumor necrosis factor receptor superfamily member 27 Proteins 0.000 description 1
- 101000679857 Homo sapiens Tumor necrosis factor receptor superfamily member 3 Proteins 0.000 description 1
- 101000606129 Homo sapiens Tyrosine-protein kinase receptor TYRO3 Proteins 0.000 description 1
- 101000855019 Homo sapiens WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 Proteins 0.000 description 1
- 101000803329 Homo sapiens WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 Proteins 0.000 description 1
- 101000666502 Homo sapiens Xaa-Pro aminopeptidase 1 Proteins 0.000 description 1
- 101000626703 Homo sapiens YEATS domain-containing protein 2 Proteins 0.000 description 1
- 101000760275 Homo sapiens Zinc finger protein 746 Proteins 0.000 description 1
- 101000818522 Homo sapiens fMet-Leu-Phe receptor Proteins 0.000 description 1
- 101000654277 Hottentotta tamulus Neurotoxin-2 Proteins 0.000 description 1
- 241000243251 Hydra Species 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000026633 IL6 Human genes 0.000 description 1
- 108091058560 IL8 Proteins 0.000 description 1
- MPBVHIBUJCELCL-UHFFFAOYSA-N Ibandronate Chemical compound CCCCCN(C)CCC(O)(P(O)(O)=O)P(O)(O)=O MPBVHIBUJCELCL-UHFFFAOYSA-N 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 108010073816 IgE Receptors Proteins 0.000 description 1
- 102000009490 IgG Receptors Human genes 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
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 241000289658 Insectivora Species 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000018682 Interleukin Receptor Common gamma Subunit Human genes 0.000 description 1
- 108010066719 Interleukin Receptor Common gamma Subunit Proteins 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 102100039064 Interleukin-3 Human genes 0.000 description 1
- 102100033502 Interleukin-37 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 102000000743 Interleukin-5 Human genes 0.000 description 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 1
- 102100037795 Interleukin-6 receptor subunit beta Human genes 0.000 description 1
- 101710152369 Interleukin-6 receptor subunit beta Proteins 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 102100026236 Interleukin-8 Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 108010056045 K cadherin Proteins 0.000 description 1
- 102100037645 Kelch domain-containing protein 10 Human genes 0.000 description 1
- 102100040445 Keratin, type I cytoskeletal 14 Human genes 0.000 description 1
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 1
- 101710194922 Keratin, type II cytoskeletal 1 Proteins 0.000 description 1
- 102100025759 Keratin, type II cytoskeletal 3 Human genes 0.000 description 1
- 102100023972 Keratin, type II cytoskeletal 8 Human genes 0.000 description 1
- 108010066321 Keratin-14 Proteins 0.000 description 1
- 108010066302 Keratin-19 Proteins 0.000 description 1
- 102000005714 Keratin-2 Human genes 0.000 description 1
- 108010070520 Keratin-2 Proteins 0.000 description 1
- 108010070918 Keratin-3 Proteins 0.000 description 1
- 108010070511 Keratin-8 Proteins 0.000 description 1
- 102100035792 Kininogen-1 Human genes 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- STECJAGHUSJQJN-USLFZFAMSA-N LSM-4015 Chemical compound C1([C@@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-USLFZFAMSA-N 0.000 description 1
- JLERVPBPJHKRBJ-UHFFFAOYSA-N LY 117018 Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCC3)=CC=2)C2=CC=C(O)C=C2S1 JLERVPBPJHKRBJ-UHFFFAOYSA-N 0.000 description 1
- 108010054278 Lac Repressors Proteins 0.000 description 1
- 229920001491 Lentinan Polymers 0.000 description 1
- 102100028919 Leucine-rich repeat transmembrane protein FLRT1 Human genes 0.000 description 1
- 102100040899 Leucine-rich repeat transmembrane protein FLRT2 Human genes 0.000 description 1
- 102100040900 Leucine-rich repeat transmembrane protein FLRT3 Human genes 0.000 description 1
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 description 1
- 102100039564 Leukosialin Human genes 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 1
- 101710106949 Long neurotoxin 1 Proteins 0.000 description 1
- 102100023111 Long-chain fatty acid transport protein 1 Human genes 0.000 description 1
- 102100023113 Long-chain fatty acid transport protein 4 Human genes 0.000 description 1
- 102100031784 Loricrin Human genes 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 102100029205 Low affinity immunoglobulin gamma Fc region receptor II-b Human genes 0.000 description 1
- 102100029206 Low affinity immunoglobulin gamma Fc region receptor II-c Human genes 0.000 description 1
- 101710099301 Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 102100033486 Lymphocyte antigen 75 Human genes 0.000 description 1
- 101710157884 Lymphocyte antigen 75 Proteins 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 102100023326 M-phase inducer phosphatase 1 Human genes 0.000 description 1
- 102100023325 M-phase inducer phosphatase 2 Human genes 0.000 description 1
- 101150104297 MASP1 gene Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 108010059343 MM Form Creatine Kinase Proteins 0.000 description 1
- 102100025136 Macrosialin Human genes 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- VJRAUFKOOPNFIQ-UHFFFAOYSA-N Marcellomycin Natural products C12=C(O)C=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C=C2C(C(=O)OC)C(CC)(O)CC1OC(OC1C)CC(N(C)C)C1OC(OC1C)CC(O)C1OC1CC(O)C(O)C(C)O1 VJRAUFKOOPNFIQ-UHFFFAOYSA-N 0.000 description 1
- 229930126263 Maytansine Natural products 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 108010090306 Member 2 Subfamily G ATP Binding Cassette Transporter Proteins 0.000 description 1
- 102100039373 Membrane cofactor protein Human genes 0.000 description 1
- IVDYZAAPOLNZKG-KWHRADDSSA-N Mepitiostane Chemical compound O([C@@H]1[C@]2(CC[C@@H]3[C@@]4(C)C[C@H]5S[C@H]5C[C@@H]4CC[C@H]3[C@@H]2CC1)C)C1(OC)CCCC1 IVDYZAAPOLNZKG-KWHRADDSSA-N 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 101710111879 Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 102300056403 Methyl-CpG-binding domain protein 2 isoform 1 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- VFKZTMPDYBFSTM-KVTDHHQDSA-N Mitobronitol Chemical compound BrC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CBr VFKZTMPDYBFSTM-KVTDHHQDSA-N 0.000 description 1
- 102100037801 Mitogen-activated protein kinase 6 Human genes 0.000 description 1
- 102100033127 Mitogen-activated protein kinase kinase kinase 5 Human genes 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101000970577 Mus musculus Bcl-2-interacting killer Proteins 0.000 description 1
- 101100381649 Mus musculus Bik gene Proteins 0.000 description 1
- 101100381845 Mus musculus Blk gene Proteins 0.000 description 1
- 101100381978 Mus musculus Braf gene Proteins 0.000 description 1
- 101000715501 Mus musculus Cathepsin 7 Proteins 0.000 description 1
- 101100167135 Mus musculus Chil3 gene Proteins 0.000 description 1
- 101000906941 Mus musculus Chitinase-like protein 3 Proteins 0.000 description 1
- 101100225547 Mus musculus Ehmt2 gene Proteins 0.000 description 1
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 1
- 101100447658 Mus musculus Gas1 gene Proteins 0.000 description 1
- 101100013973 Mus musculus Gata4 gene Proteins 0.000 description 1
- 101100125281 Mus musculus Hoxd11 gene Proteins 0.000 description 1
- 101100236114 Mus musculus Lrrfip1 gene Proteins 0.000 description 1
- 101001065556 Mus musculus Lymphocyte antigen 6G Proteins 0.000 description 1
- 101100369076 Mus musculus Tdgf1 gene Proteins 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 108010056852 Myostatin Proteins 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 108010028286 N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase Proteins 0.000 description 1
- 108050000637 N-cadherin Proteins 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 102100021126 N-formyl peptide receptor 2 Human genes 0.000 description 1
- 102100028130 N-formyl peptide receptor 3 Human genes 0.000 description 1
- 101000783409 Naja anchietae Long neurotoxin 1 Proteins 0.000 description 1
- 101000744155 Naja atra Cytotoxin 3 Proteins 0.000 description 1
- 101000800759 Naja mossambica Short neurotoxin 1 Proteins 0.000 description 1
- 101000800755 Naja oxiana Alpha-elapitoxin-Nno2a Proteins 0.000 description 1
- 101000800760 Naja oxiana Short neurotoxin 1 Proteins 0.000 description 1
- 102100038082 Natural killer cell receptor 2B4 Human genes 0.000 description 1
- 101710141230 Natural killer cell receptor 2B4 Proteins 0.000 description 1
- 102100021462 Natural killer cells antigen CD94 Human genes 0.000 description 1
- 239000005104 Neeliglow 4-amino-1,8-naphthalimide Substances 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102100023206 Neuromodulin Human genes 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- SYNHCENRCUAUNM-UHFFFAOYSA-N Nitrogen mustard N-oxide hydrochloride Chemical compound Cl.ClCC[N+]([O-])(C)CCCl SYNHCENRCUAUNM-UHFFFAOYSA-N 0.000 description 1
- KGTDRFCXGRULNK-UHFFFAOYSA-N Nogalamycin Natural products COC1C(OC)(C)C(OC)C(C)OC1OC1C2=C(O)C(C(=O)C3=C(O)C=C4C5(C)OC(C(C(C5O)N(C)C)O)OC4=C3C3=O)=C3C=C2C(C(=O)OC)C(C)(O)C1 KGTDRFCXGRULNK-UHFFFAOYSA-N 0.000 description 1
- 102000005650 Notch Receptors Human genes 0.000 description 1
- 108010070047 Notch Receptors Proteins 0.000 description 1
- 102100038512 Nuclear receptor subfamily 1 group I member 3 Human genes 0.000 description 1
- 101710093927 Nuclear receptor subfamily 6 group A member 1 Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108010042215 OX40 Ligand Proteins 0.000 description 1
- 229930187135 Olivomycin Natural products 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108010084438 Oncogene Protein v-maf Proteins 0.000 description 1
- 102100031943 One cut domain family member 2 Human genes 0.000 description 1
- 102000003840 Opioid Receptors Human genes 0.000 description 1
- 108090000137 Opioid Receptors Proteins 0.000 description 1
- 108010061952 Orosomucoid Proteins 0.000 description 1
- 102000012404 Orosomucoid Human genes 0.000 description 1
- 101100381429 Oryza sativa subsp. japonica BADH2 gene Proteins 0.000 description 1
- 102000004264 Osteopontin Human genes 0.000 description 1
- 108010081689 Osteopontin Proteins 0.000 description 1
- 108010035042 Osteoprotegerin Proteins 0.000 description 1
- 102100026459 POU domain, class 3, transcription factor 2 Human genes 0.000 description 1
- 101710133394 POU domain, class 3, transcription factor 2 Proteins 0.000 description 1
- 102100026456 POU domain, class 3, transcription factor 3 Human genes 0.000 description 1
- 101710133393 POU domain, class 3, transcription factor 3 Proteins 0.000 description 1
- 102100035395 POU domain, class 4, transcription factor 1 Human genes 0.000 description 1
- 102100024894 PR domain zinc finger protein 1 Human genes 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 208000016222 Pancreatic disease Diseases 0.000 description 1
- 241000051107 Paraechinus aethiopicus Species 0.000 description 1
- 108010057150 Peplomycin Proteins 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 102100032393 Peptidoglycan recognition protein 1 Human genes 0.000 description 1
- 101710113134 Peptidoglycan recognition protein 1 Proteins 0.000 description 1
- 102100034539 Peptidyl-prolyl cis-trans isomerase A Human genes 0.000 description 1
- 102100036978 Peptidyl-prolyl cis-trans isomerase FKBP8 Human genes 0.000 description 1
- KMSKQZKKOZQFFG-HSUXVGOQSA-N Pirarubicin Chemical compound O([C@H]1[C@@H](N)C[C@@H](O[C@H]1C)O[C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1CCCCO1 KMSKQZKKOZQFFG-HSUXVGOQSA-N 0.000 description 1
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 102100038394 Platelet glycoprotein VI Human genes 0.000 description 1
- 101710194982 Platelet glycoprotein VI Proteins 0.000 description 1
- 101100335198 Pneumocystis carinii fol1 gene Proteins 0.000 description 1
- 102100024588 Podocalyxin-like protein 2 Human genes 0.000 description 1
- 108091010857 Podocalyxin-like protein 2 Proteins 0.000 description 1
- 102100023715 Poly(A)-specific ribonuclease PARN Human genes 0.000 description 1
- 229920000805 Polyaspartic acid Polymers 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 241000702619 Porcine parvovirus Species 0.000 description 1
- 108010009975 Positive Regulatory Domain I-Binding Factor 1 Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102000004257 Potassium Channel Human genes 0.000 description 1
- HFVNWDWLWUCIHC-GUPDPFMOSA-N Prednimustine Chemical compound O=C([C@@]1(O)CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)[C@@H](O)C[C@@]21C)COC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 HFVNWDWLWUCIHC-GUPDPFMOSA-N 0.000 description 1
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102100025974 Pro-cathepsin H Human genes 0.000 description 1
- 102100040918 Pro-glucagon Human genes 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 101710092628 Probable histidine-binding protein Proteins 0.000 description 1
- 102100029837 Probetacellulin Human genes 0.000 description 1
- 102100025498 Proepiregulin Human genes 0.000 description 1
- 102100040126 Prokineticin-1 Human genes 0.000 description 1
- 102100038277 Prostaglandin G/H synthase 1 Human genes 0.000 description 1
- 108050003243 Prostaglandin G/H synthase 1 Proteins 0.000 description 1
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 102000017975 Protein C Human genes 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102100027796 Protein CREG1 Human genes 0.000 description 1
- 102100040819 Protein FAM3A Human genes 0.000 description 1
- 102100040307 Protein FAM3B Human genes 0.000 description 1
- 102100040823 Protein FAM3C Human genes 0.000 description 1
- 102100040821 Protein FAM3D Human genes 0.000 description 1
- 102100026734 Protein FAM98A Human genes 0.000 description 1
- 102100020847 Protein FosB Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108091008611 Protein Kinase B Proteins 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 101710110949 Protein S100-A12 Proteins 0.000 description 1
- 102100027097 Protein amnionless Human genes 0.000 description 1
- 102100035622 Protein dispatched homolog 1 Human genes 0.000 description 1
- 102100029371 Protein disulfide isomerase CRELD1 Human genes 0.000 description 1
- 102100029370 Protein disulfide isomerase CRELD2 Human genes 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 108010001859 Proto-Oncogene Proteins c-rel Proteins 0.000 description 1
- 102000000850 Proto-Oncogene Proteins c-rel Human genes 0.000 description 1
- 101000762949 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) Exotoxin A Proteins 0.000 description 1
- 108010025832 RANK Ligand Proteins 0.000 description 1
- 102000018795 RELT Human genes 0.000 description 1
- 108010052562 RELT Proteins 0.000 description 1
- 102100025873 RNA-binding protein 27 Human genes 0.000 description 1
- 101710206013 RNA-binding protein 27 Proteins 0.000 description 1
- AHHFEZNOXOZZQA-ZEBDFXRSSA-N Ranimustine Chemical compound CO[C@H]1O[C@H](CNC(=O)N(CCCl)N=O)[C@@H](O)[C@H](O)[C@H]1O AHHFEZNOXOZZQA-ZEBDFXRSSA-N 0.000 description 1
- 102100031429 Ras-interacting protein 1 Human genes 0.000 description 1
- 101710133005 Ras-interacting protein 1 Proteins 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 101000947192 Rattus norvegicus C-X-C motif chemokine 2 Proteins 0.000 description 1
- 101000947185 Rattus norvegicus C-X-C motif chemokine 3 Proteins 0.000 description 1
- 101000899441 Rattus norvegicus Cadherin-22 Proteins 0.000 description 1
- 101001069900 Rattus norvegicus Growth-regulated alpha protein Proteins 0.000 description 1
- 108010038036 Receptor Activator of Nuclear Factor-kappa B Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 201000007737 Retinal degeneration Diseases 0.000 description 1
- 102100033914 Retinoic acid receptor responder protein 2 Human genes 0.000 description 1
- 101710170513 Retinoic acid receptor responder protein 2 Proteins 0.000 description 1
- 241000282798 Rhinocerotidae Species 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101150058068 SLC2A1 gene Proteins 0.000 description 1
- 101150052594 SLC2A3 gene Proteins 0.000 description 1
- 101150110386 SLC2A4 gene Proteins 0.000 description 1
- 101150049961 SLC2A5 gene Proteins 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- 102100030058 Secreted frizzled-related protein 1 Human genes 0.000 description 1
- 108050007993 Secreted frizzled-related protein 1 Proteins 0.000 description 1
- 108050007987 Secreted frizzled-related protein 2 Proteins 0.000 description 1
- 102100030054 Secreted frizzled-related protein 2 Human genes 0.000 description 1
- 108050007990 Secreted frizzled-related protein 3 Proteins 0.000 description 1
- 102100030053 Secreted frizzled-related protein 3 Human genes 0.000 description 1
- 102100030052 Secreted frizzled-related protein 4 Human genes 0.000 description 1
- 108050008088 Secreted frizzled-related protein 4 Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102100035719 Serine/arginine-rich splicing factor 11 Human genes 0.000 description 1
- 102100028949 Serine/threonine-protein kinase TAO2 Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108700000090 Sfrs11 Proteins 0.000 description 1
- 102100027318 Signal recognition particle subunit SRP68 Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920000519 Sizofiran Polymers 0.000 description 1
- 101150116689 Slc2a2 gene Proteins 0.000 description 1
- 102100037446 Small conductance calcium-activated potassium channel protein 2 Human genes 0.000 description 1
- 101710201182 Small conductance calcium-activated potassium channel protein 2 Proteins 0.000 description 1
- 101710173239 Small nuclear ribonucleoprotein Sm D1 Proteins 0.000 description 1
- 101000690440 Solanum lycopersicum Floral homeotic protein AGAMOUS Proteins 0.000 description 1
- 102100022831 Somatoliberin Human genes 0.000 description 1
- 101710142969 Somatoliberin Proteins 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 241000288726 Soricidae Species 0.000 description 1
- 102300036221 Sperm-associated antigen 8 isoform 1 Human genes 0.000 description 1
- 102300036219 Sperm-associated antigen 8 isoform 2 Human genes 0.000 description 1
- 102100025750 Sphingosine 1-phosphate receptor 1 Human genes 0.000 description 1
- 102100029802 Sphingosine 1-phosphate receptor 5 Human genes 0.000 description 1
- 101710145796 Staphylokinase Proteins 0.000 description 1
- 102100036832 Steroid hormone receptor ERR1 Human genes 0.000 description 1
- 102100036831 Steroid hormone receptor ERR2 Human genes 0.000 description 1
- 108010087999 Steryl-Sulfatase Proteins 0.000 description 1
- 101000588932 Stichodactyla helianthus Delta-stichotoxin-She1a Proteins 0.000 description 1
- 241000201081 Streptomyces maritimus Species 0.000 description 1
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 1
- 108010090618 Syntaxin 1 Proteins 0.000 description 1
- 108700038219 Syntaxin-2 Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 102100025131 T-cell differentiation antigen CD6 Human genes 0.000 description 1
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 1
- 101710114141 T-lymphocyte surface antigen Ly-9 Proteins 0.000 description 1
- 108010076818 TEV protease Proteins 0.000 description 1
- 102100033455 TGF-beta receptor type-2 Human genes 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 108091007178 TNFRSF10A Proteins 0.000 description 1
- 102100027802 Target of rapamycin complex subunit LST8 Human genes 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- CGMTUJFWROPELF-UHFFFAOYSA-N Tenuazonic acid Natural products CCC(C)C1NC(=O)C(=C(C)/O)C1=O CGMTUJFWROPELF-UHFFFAOYSA-N 0.000 description 1
- 108010049264 Teriparatide Proteins 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 1
- 101150116166 Thbs3 gene Proteins 0.000 description 1
- 102100034196 Thrombopoietin receptor Human genes 0.000 description 1
- 101710148535 Thrombopoietin receptor Proteins 0.000 description 1
- 102100036034 Thrombospondin-1 Human genes 0.000 description 1
- 102100029529 Thrombospondin-2 Human genes 0.000 description 1
- 102100029524 Thrombospondin-3 Human genes 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 102100033504 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000011923 Thyrotropin Human genes 0.000 description 1
- 108010061174 Thyrotropin Proteins 0.000 description 1
- IWEQQRMGNVVKQW-OQKDUQJOSA-N Toremifene citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 IWEQQRMGNVVKQW-OQKDUQJOSA-N 0.000 description 1
- 101710182223 Toxin B Proteins 0.000 description 1
- CMCSZZOVEJFBEY-UHFFFAOYSA-N Toxin FS2 Chemical compound C1CC(C)(O)C=CC1(C)C1(C)CC(O)C=C1CO CMCSZZOVEJFBEY-UHFFFAOYSA-N 0.000 description 1
- 108010063395 Transcription Factor Brn-3A Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102300049659 Transcription factor 20 isoform 1 Human genes 0.000 description 1
- 102300049667 Transcription factor 20 isoform 2 Human genes 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 108050004365 Transcription factor Maf Proteins 0.000 description 1
- 108050008750 Transcription factor SOX-1 Proteins 0.000 description 1
- 101710171413 Transcriptional repressor scratch 2 Proteins 0.000 description 1
- 102100039362 Transducin-like enhancer protein 1 Human genes 0.000 description 1
- 101710101305 Transducin-like enhancer protein 1 Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 102000007641 Trefoil Factors Human genes 0.000 description 1
- 108010007389 Trefoil Factors Proteins 0.000 description 1
- UMILHIMHKXVDGH-UHFFFAOYSA-N Triethylene glycol diglycidyl ether Chemical compound C1OC1COCCOCCOCCOCC1CO1 UMILHIMHKXVDGH-UHFFFAOYSA-N 0.000 description 1
- 235000015724 Trifolium pratense Nutrition 0.000 description 1
- 102100028101 Triple functional domain protein Human genes 0.000 description 1
- 102100040653 Tryptophan 2,3-dioxygenase Human genes 0.000 description 1
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 1
- 102000012883 Tumor Necrosis Factor Ligand Superfamily Member 14 Human genes 0.000 description 1
- 108010065158 Tumor Necrosis Factor Ligand Superfamily Member 14 Proteins 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 102000000504 Tumor Suppressor p53-Binding Protein 1 Human genes 0.000 description 1
- 108090000138 Tumor necrosis factor ligand superfamily member 15 Proteins 0.000 description 1
- 102100040110 Tumor necrosis factor receptor superfamily member 10D Human genes 0.000 description 1
- 102100028787 Tumor necrosis factor receptor superfamily member 11A Human genes 0.000 description 1
- 101710178300 Tumor necrosis factor receptor superfamily member 13C Proteins 0.000 description 1
- 102100033725 Tumor necrosis factor receptor superfamily member 16 Human genes 0.000 description 1
- 102100026716 Tumor necrosis factor receptor superfamily member 19L Human genes 0.000 description 1
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 1
- 102100022202 Tumor necrosis factor receptor superfamily member 27 Human genes 0.000 description 1
- 102100022156 Tumor necrosis factor receptor superfamily member 3 Human genes 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 102000000523 Type II Activin Receptors Human genes 0.000 description 1
- 108010041546 Type II Activin Receptors Proteins 0.000 description 1
- 102100039127 Tyrosine-protein kinase receptor TYRO3 Human genes 0.000 description 1
- 101710100170 Unknown protein Proteins 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 108010079206 V-Set Domain-Containing T-Cell Activation Inhibitor 1 Proteins 0.000 description 1
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 description 1
- 102000008790 VE-cadherin Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 101710160039 WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 Proteins 0.000 description 1
- 101710160038 WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 Proteins 0.000 description 1
- 108010038900 X-Pro aminopeptidase Proteins 0.000 description 1
- 102100038365 Xaa-Pro aminopeptidase 1 Human genes 0.000 description 1
- 102100038364 Xaa-Pro aminopeptidase 2 Human genes 0.000 description 1
- 101001074046 Xenopus laevis Zinc finger protein GLI1 Proteins 0.000 description 1
- 101001029301 Xenopus tropicalis Forkhead box protein D3 Proteins 0.000 description 1
- 101001023436 Xenopus tropicalis Forkhead box protein J1 Proteins 0.000 description 1
- 102100024781 YEATS domain-containing protein 2 Human genes 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 102100026316 Zinc finger protein 281 Human genes 0.000 description 1
- 101710143983 Zinc finger protein 281 Proteins 0.000 description 1
- 102100024724 Zinc finger protein 579 Human genes 0.000 description 1
- 101710143321 Zinc finger protein 579 Proteins 0.000 description 1
- 102100024714 Zinc finger protein 746 Human genes 0.000 description 1
- IZOBIZVXEKNCNN-ZNQIEUMMSA-N [(1r,2r,3's,4e,5s)-4-hexa-2,4-diynylidenespiro[3,6-dioxabicyclo[3.1.0]hexane-2,6'-oxane]-3'-yl] 3-methylbutanoate Chemical compound CC#CC#C\C=C([C@H]1O[C@H]11)\O[C@@]21CC[C@H](OC(=O)CC(C)C)CO2 IZOBIZVXEKNCNN-ZNQIEUMMSA-N 0.000 description 1
- ZYVSOIYQKUDENJ-ASUJBHBQSA-N [(2R,3R,4R,6R)-6-[[(6S,7S)-6-[(2S,4R,5R,6R)-4-[(2R,4R,5R,6R)-4-[(2S,4S,5S,6S)-5-acetyloxy-4-hydroxy-4,6-dimethyloxan-2-yl]oxy-5-hydroxy-6-methyloxan-2-yl]oxy-5-hydroxy-6-methyloxan-2-yl]oxy-7-[(3S,4R)-3,4-dihydroxy-1-methoxy-2-oxopentyl]-4,10-dihydroxy-3-methyl-5-oxo-7,8-dihydro-6H-anthracen-2-yl]oxy]-4-[(2R,4R,5R,6R)-4-hydroxy-5-methoxy-6-methyloxan-2-yl]oxy-2-methyloxan-3-yl] acetate Chemical class COC([C@@H]1Cc2cc3cc(O[C@@H]4C[C@@H](O[C@@H]5C[C@@H](O)[C@@H](OC)[C@@H](C)O5)[C@H](OC(C)=O)[C@@H](C)O4)c(C)c(O)c3c(O)c2C(=O)[C@H]1O[C@H]1C[C@@H](O[C@@H]2C[C@@H](O[C@H]3C[C@](C)(O)[C@@H](OC(C)=O)[C@H](C)O3)[C@H](O)[C@@H](C)O2)[C@H](O)[C@@H](C)O1)C(=O)[C@@H](O)[C@@H](C)O ZYVSOIYQKUDENJ-ASUJBHBQSA-N 0.000 description 1
- SPJCRMJCFSJKDE-ZWBUGVOYSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] 2-[4-[bis(2-chloroethyl)amino]phenyl]acetate Chemical compound O([C@@H]1CC2=CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CCCC(C)C)C(=O)CC1=CC=C(N(CCCl)CCCl)C=C1 SPJCRMJCFSJKDE-ZWBUGVOYSA-N 0.000 description 1
- IFJUINDAXYAPTO-UUBSBJJBSA-N [(8r,9s,13s,14s,17s)-17-[2-[4-[4-[bis(2-chloroethyl)amino]phenyl]butanoyloxy]acetyl]oxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate Chemical compound C([C@@H]1[C@@H](C2=CC=3)CC[C@]4([C@H]1CC[C@@H]4OC(=O)COC(=O)CCCC=1C=CC(=CC=1)N(CCCl)CCCl)C)CC2=CC=3OC(=O)C1=CC=CC=C1 IFJUINDAXYAPTO-UUBSBJJBSA-N 0.000 description 1
- IHGLINDYFMDHJG-UHFFFAOYSA-N [2-(4-methoxyphenyl)-3,4-dihydronaphthalen-1-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]methanone Chemical compound C1=CC(OC)=CC=C1C(CCC1=CC=CC=C11)=C1C(=O)C(C=C1)=CC=C1OCCN1CCCC1 IHGLINDYFMDHJG-UHFFFAOYSA-N 0.000 description 1
- XZSRRNFBEIOBDA-CFNBKWCHSA-N [2-[(2s,4s)-4-[(2r,4s,5s,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1h-tetracen-2-yl]-2-oxoethyl] 2,2-diethoxyacetate Chemical compound O([C@H]1C[C@](CC2=C(O)C=3C(=O)C4=CC=CC(OC)=C4C(=O)C=3C(O)=C21)(O)C(=O)COC(=O)C(OCC)OCC)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 XZSRRNFBEIOBDA-CFNBKWCHSA-N 0.000 description 1
- ZOZKYEHVNDEUCO-XUTVFYLZSA-N aceglatone Chemical compound O1C(=O)[C@H](OC(C)=O)[C@@H]2OC(=O)[C@@H](OC(=O)C)[C@@H]21 ZOZKYEHVNDEUCO-XUTVFYLZSA-N 0.000 description 1
- 229950002684 aceglatone Drugs 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229930183665 actinomycin Natural products 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 108010023082 activin A Proteins 0.000 description 1
- 108010023079 activin B Proteins 0.000 description 1
- 229940060205 adagen Drugs 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000000808 adrenergic beta-agonist Substances 0.000 description 1
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 101150045355 akt1 gene Proteins 0.000 description 1
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 108010018360 alpha 2u globulin Proteins 0.000 description 1
- 239000002776 alpha toxin Substances 0.000 description 1
- 102000012005 alpha-2-HS-Glycoprotein Human genes 0.000 description 1
- 108010075843 alpha-2-HS-Glycoprotein Proteins 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 1
- 108010055359 alpha-cobratoxin Proteins 0.000 description 1
- 229960000473 altretamine Drugs 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 108010003977 aminoacylase I Proteins 0.000 description 1
- 229960003437 aminoglutethimide Drugs 0.000 description 1
- ROBVIMPUHSLWNV-UHFFFAOYSA-N aminoglutethimide Chemical compound C=1C=C(N)C=CC=1C1(CC)CCC(=O)NC1=O ROBVIMPUHSLWNV-UHFFFAOYSA-N 0.000 description 1
- 229960002749 aminolevulinic acid Drugs 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229960001220 amsacrine Drugs 0.000 description 1
- XCPGHVQEEXUHNC-UHFFFAOYSA-N amsacrine Chemical compound COC1=CC(NS(C)(=O)=O)=CC=C1NC1=C(C=CC=C2)C2=NC2=CC=CC=C12 XCPGHVQEEXUHNC-UHFFFAOYSA-N 0.000 description 1
- 238000012435 analytical chromatography Methods 0.000 description 1
- BBDAGFIXKZCXAH-CCXZUQQUSA-N ancitabine Chemical compound N=C1C=CN2[C@@H]3O[C@H](CO)[C@@H](O)[C@@H]3OC2=N1 BBDAGFIXKZCXAH-CCXZUQQUSA-N 0.000 description 1
- 229950000242 ancitabine Drugs 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 208000020731 anterior segment dysgenesis 3 Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002280 anti-androgenic effect Effects 0.000 description 1
- 230000003474 anti-emetic effect Effects 0.000 description 1
- 229940046836 anti-estrogen Drugs 0.000 description 1
- 230000001833 anti-estrogenic effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002303 anti-venom Effects 0.000 description 1
- 239000000051 antiandrogen Substances 0.000 description 1
- 229940030495 antiandrogen sex hormone and modulator of the genital system Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 229940125683 antiemetic agent Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000013059 antihormonal agent Substances 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 229940045687 antimetabolites folic acid analogs Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 229940125716 antipyretic agent Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000013011 aqueous formulation Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 150000008209 arabinosides Chemical class 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 150000001508 asparagines Chemical class 0.000 description 1
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 1
- 108010044540 auristatin Proteins 0.000 description 1
- 239000005441 aurora Substances 0.000 description 1
- 108010014210 axokine Proteins 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 1
- 229950011321 azaserine Drugs 0.000 description 1
- 150000001541 aziridines Chemical class 0.000 description 1
- 238000002819 bacterial display Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 102000055102 bcl-2-Associated X Human genes 0.000 description 1
- 108700000707 bcl-2-Associated X Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DQEPMTIXHXSFOR-UHFFFAOYSA-N benzo[a]pyrene diol epoxide I Chemical compound C1=C2C(C3OC3C(C3O)O)=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 DQEPMTIXHXSFOR-UHFFFAOYSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 108700006666 betaIG-H3 Proteins 0.000 description 1
- 229960000997 bicalutamide Drugs 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 229950008548 bisantrene Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical class N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
- 239000003152 bradykinin antagonist Substances 0.000 description 1
- 229960003150 bupivacaine Drugs 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 108700002839 cactinomycin Proteins 0.000 description 1
- 229950009908 cactinomycin Drugs 0.000 description 1
- 108010018828 cadherin 5 Proteins 0.000 description 1
- 102000014823 calbindin Human genes 0.000 description 1
- 108060001061 calbindin Proteins 0.000 description 1
- 239000000480 calcium channel blocker Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229930195731 calicheamicin Natural products 0.000 description 1
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 description 1
- 229950009823 calusterone Drugs 0.000 description 1
- IVFYLRMMHVYGJH-PVPPCFLZSA-N calusterone Chemical compound C1C[C@]2(C)[C@](O)(C)CC[C@H]2[C@@H]2[C@@H](C)CC3=CC(=O)CC[C@]3(C)[C@H]21 IVFYLRMMHVYGJH-PVPPCFLZSA-N 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 108010019521 carbonic anhydrase VI Proteins 0.000 description 1
- 108010087312 carbonic anhydrase XII Proteins 0.000 description 1
- 108010087372 carbonic anhydrase XIV Proteins 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 229960002115 carboquone Drugs 0.000 description 1
- 108010011091 cardiotoxin V Proteins 0.000 description 1
- GCDJGEWSHKXMFB-KLSUCABJSA-N cardiotoxin, cobra Chemical compound C([C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@H]2CSSC[C@H](NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]3CCCN3C(=O)[C@H](C(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]3N(CCC3)C2=O)CSSC[C@@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N2CCC[C@H]2C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N1)C(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]1C(N2CCC[C@H]2C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@H](C(=O)N[C@@H](CSSC1)C(=O)N[C@@H]1C(N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CSSC1)C(=O)N[C@@H](CC(N)=O)C(O)=O)[C@@H](C)O)=O)C(C)C)C(C)C)=O)[C@@H](C)O)C1=CC=C(O)C=C1 GCDJGEWSHKXMFB-KLSUCABJSA-N 0.000 description 1
- 108010041776 cardiotrophin 1 Proteins 0.000 description 1
- 229960003261 carmofur Drugs 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 108010047060 carzinophilin Proteins 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- 108010018550 caspase 13 Proteins 0.000 description 1
- 108010033380 cathepsin-6 Proteins 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000004656 cell transport Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 101150113535 chek1 gene Proteins 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 229910052729 chemical element Inorganic materials 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000012993 chemical processing Methods 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 108010057052 chitotriosidase Proteins 0.000 description 1
- 229950008249 chlornaphazine Drugs 0.000 description 1
- BFPSDSIWYFKGBC-UHFFFAOYSA-N chlorotrianisene Chemical compound C1=CC(OC)=CC=C1C(Cl)=C(C=1C=CC(OC)=CC=1)C1=CC=C(OC)C=C1 BFPSDSIWYFKGBC-UHFFFAOYSA-N 0.000 description 1
- 229960001480 chlorozotocin Drugs 0.000 description 1
- 102000006533 chordin Human genes 0.000 description 1
- 108010008846 chordin Proteins 0.000 description 1
- 238000011098 chromatofocusing Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000000978 circular dichroism spectroscopy Methods 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 108010072917 class-I restricted T cell-associated molecule Proteins 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 108010055222 clotting enzyme Proteins 0.000 description 1
- 229940105772 coagulation factor vii Drugs 0.000 description 1
- 229940105756 coagulation factor x Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 108010045512 cohesins Proteins 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000001054 cortical effect Effects 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
- 229940109239 creatinine Drugs 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- ZOOGRGPOEVQQDX-UHFFFAOYSA-N cyclic GMP Natural products O1C2COP(O)(=O)OC2C(O)C1N1C=NC2=C1NC(N)=NC2=O ZOOGRGPOEVQQDX-UHFFFAOYSA-N 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 108010025838 dectin 1 Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 229960005052 demecolcine Drugs 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 210000005045 desmin Anatomy 0.000 description 1
- 229950001282 desmoteplase Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229950003913 detorubicin Drugs 0.000 description 1
- LSXWFXONGKSEMY-UHFFFAOYSA-N di-tert-butyl peroxide Chemical compound CC(C)(C)OOC(C)(C)C LSXWFXONGKSEMY-UHFFFAOYSA-N 0.000 description 1
- 239000012969 di-tertiary-butyl peroxide Substances 0.000 description 1
- WVYXNIXAMZOZFK-UHFFFAOYSA-N diaziquone Chemical compound O=C1C(NC(=O)OCC)=C(N2CC2)C(=O)C(NC(=O)OCC)=C1N1CC1 WVYXNIXAMZOZFK-UHFFFAOYSA-N 0.000 description 1
- 229950002389 diaziquone Drugs 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 230000000447 dimerizing effect Effects 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- ZWAOHEXOSAUJHY-ZIYNGMLESA-N doxifluridine Chemical compound O[C@@H]1[C@H](O)[C@@H](C)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ZWAOHEXOSAUJHY-ZIYNGMLESA-N 0.000 description 1
- 229950005454 doxifluridine Drugs 0.000 description 1
- NOTIQUSPUUHHEH-UXOVVSIBSA-N dromostanolone propionate Chemical compound C([C@@H]1CC2)C(=O)[C@H](C)C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](OC(=O)CC)[C@@]2(C)CC1 NOTIQUSPUUHHEH-UXOVVSIBSA-N 0.000 description 1
- 229950004683 drostanolone propionate Drugs 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- VLCYCQAOQCDTCN-UHFFFAOYSA-N eflornithine Chemical compound NCCCC(N)(C(F)F)C(O)=O VLCYCQAOQCDTCN-UHFFFAOYSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229950000549 elliptinium acetate Drugs 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229950011487 enocitabine Drugs 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 229950002973 epitiostanol Drugs 0.000 description 1
- 108010033998 erabutoxin A Proteins 0.000 description 1
- SRWAKURFIXSMSY-YIPRCOCFSA-N erabutoxin a Chemical compound C([C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CSSC[C@H]2C(=O)N3CCC[C@H]3C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=3C4=CC=CC=C4NC=3)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N1)=O)CSSC[C@@H](C(N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N2)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)NCC(=O)N[C@@H]1C(N2CCC[C@H]2C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N2CCC[C@H]2C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CSSC1)C(=O)N[C@@H]1C(N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(=O)N[C@@H](CSSC1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)C(C)C)=O)[C@@H](C)CC)C(C)C)[C@@H](C)O)=O)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)C1=CC=CC=C1 SRWAKURFIXSMSY-YIPRCOCFSA-N 0.000 description 1
- WITZDNDAZYTMTC-MJQGTENLSA-N erabutoxin b Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H]([C@@H](C)CC)NC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]1N(C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=2NC=NC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC=2NC=NC=2)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)CCC1 WITZDNDAZYTMTC-MJQGTENLSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229950002017 esorubicin Drugs 0.000 description 1
- ITSGNOIFAJAQHJ-BMFNZSJVSA-N esorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)C[C@H](C)O1 ITSGNOIFAJAQHJ-BMFNZSJVSA-N 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- 108091008559 estrogen-related receptor alpha Proteins 0.000 description 1
- 108091008558 estrogen-related receptor beta Proteins 0.000 description 1
- 108091008557 estrogen-related receptor gamma Proteins 0.000 description 1
- QSRLNKCNOLVZIR-KRWDZBQOSA-N ethyl (2s)-2-[[2-[4-[bis(2-chloroethyl)amino]phenyl]acetyl]amino]-4-methylsulfanylbutanoate Chemical compound CCOC(=O)[C@H](CCSC)NC(=O)CC1=CC=C(N(CCCl)CCCl)C=C1 QSRLNKCNOLVZIR-KRWDZBQOSA-N 0.000 description 1
- IYBKWXQWKPSYDT-UHFFFAOYSA-L ethylene glycol disuccinate bis(sulfo-N-succinimidyl) ester sodium salt Chemical compound [Na+].[Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCC(=O)OCCOC(=O)CCC(=O)ON1C(=O)C(S([O-])(=O)=O)CC1=O IYBKWXQWKPSYDT-UHFFFAOYSA-L 0.000 description 1
- YOMFVLRTMZWACQ-UHFFFAOYSA-N ethyltrimethylammonium Chemical compound CC[N+](C)(C)C YOMFVLRTMZWACQ-UHFFFAOYSA-N 0.000 description 1
- 229960005237 etoglucid Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 102100021145 fMet-Leu-Phe receptor Human genes 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 229940012426 factor x Drugs 0.000 description 1
- 229940043168 fareston Drugs 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 150000004665 fatty acids Chemical group 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 108020002231 fibrillarin Proteins 0.000 description 1
- 102000005525 fibrillarin Human genes 0.000 description 1
- 108010072257 fibroblast activation protein alpha Proteins 0.000 description 1
- 108090000047 fibroblast growth factor 13 Proteins 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- 108700014844 flt3 ligand Proteins 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- 229960002074 flutamide Drugs 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- 101150064107 fosB gene Proteins 0.000 description 1
- 229960004783 fotemustine Drugs 0.000 description 1
- YAKWPXVTIGTRJH-UHFFFAOYSA-N fotemustine Chemical compound CCOP(=O)(OCC)C(C)NC(=O)N(CCCl)N=O YAKWPXVTIGTRJH-UHFFFAOYSA-N 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229940044658 gallium nitrate Drugs 0.000 description 1
- 230000006251 gamma-carboxylation Effects 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 235000019410 glycyrrhizin Nutrition 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 229960002913 goserelin Drugs 0.000 description 1
- 238000003875 gradient-accelerated spectroscopy Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000024348 heart neoplasm Diseases 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 229920000140 heteropolymer Polymers 0.000 description 1
- UUVWYPNAQBNQJQ-UHFFFAOYSA-N hexamethylmelamine Chemical compound CN(C)C1=NC(N(C)C)=NC(N(C)C)=N1 UUVWYPNAQBNQJQ-UHFFFAOYSA-N 0.000 description 1
- 229920006158 high molecular weight polymer Polymers 0.000 description 1
- 229940006607 hirudin Drugs 0.000 description 1
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 239000003453 histamine agonist Substances 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 150000002429 hydrazines Chemical class 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 229940015872 ibandronate Drugs 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- DBIGHPPNXATHOF-UHFFFAOYSA-N improsulfan Chemical compound CS(=O)(=O)OCCCNCCCOS(C)(=O)=O DBIGHPPNXATHOF-UHFFFAOYSA-N 0.000 description 1
- 229950008097 improsulfan Drugs 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 108040006849 interleukin-2 receptor activity proteins Proteins 0.000 description 1
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000008863 intramolecular interaction Effects 0.000 description 1
- 108010003082 intrinsic factor-cobalamin receptor Proteins 0.000 description 1
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- OEXHQOGQTVQTAT-JRNQLAHRSA-N ipratropium Chemical compound O([C@H]1C[C@H]2CC[C@@H](C1)[N@@+]2(C)C(C)C)C(=O)C(CO)C1=CC=CC=C1 OEXHQOGQTVQTAT-JRNQLAHRSA-N 0.000 description 1
- 229960001888 ipratropium Drugs 0.000 description 1
- 201000008633 iridogoniodysgenesis syndrome Diseases 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 1
- 238000012804 iterative process Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229940115286 lentinan Drugs 0.000 description 1
- 108010080821 leucine-rich amelogenin peptide Proteins 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003199 leukotriene receptor blocking agent Substances 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 229960003538 lonidamine Drugs 0.000 description 1
- WDRYRZXSPDWGEB-UHFFFAOYSA-N lonidamine Chemical compound C12=CC=CC=C2C(C(=O)O)=NN1CC1=CC=C(Cl)C=C1Cl WDRYRZXSPDWGEB-UHFFFAOYSA-N 0.000 description 1
- 108010079309 loricrin Proteins 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000006674 lysosomal degradation Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- MQXVYODZCMMZEM-ZYUZMQFOSA-N mannomustine Chemical compound ClCCNC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CNCCCl MQXVYODZCMMZEM-ZYUZMQFOSA-N 0.000 description 1
- 229950008612 mannomustine Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 108010000525 member 1 small inducible cytokine subfamily E Proteins 0.000 description 1
- 229950009246 mepitiostane Drugs 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- VJRAUFKOOPNFIQ-TVEKBUMESA-N methyl (1r,2r,4s)-4-[(2r,4s,5s,6s)-5-[(2s,4s,5s,6s)-5-[(2s,4s,5s,6s)-4,5-dihydroxy-6-methyloxan-2-yl]oxy-4-hydroxy-6-methyloxan-2-yl]oxy-4-(dimethylamino)-6-methyloxan-2-yl]oxy-2-ethyl-2,5,7,10-tetrahydroxy-6,11-dioxo-3,4-dihydro-1h-tetracene-1-carboxylat Chemical compound O([C@H]1[C@@H](O)C[C@@H](O[C@H]1C)O[C@H]1[C@H](C[C@@H](O[C@H]1C)O[C@H]1C[C@]([C@@H](C2=CC=3C(=O)C4=C(O)C=CC(O)=C4C(=O)C=3C(O)=C21)C(=O)OC)(O)CC)N(C)C)[C@H]1C[C@H](O)[C@H](O)[C@H](C)O1 VJRAUFKOOPNFIQ-TVEKBUMESA-N 0.000 description 1
- CQBCVFHLZAVNPF-UHFFFAOYSA-N methyl 3-[(3-imino-3-methoxypropyl)disulfanyl]propanimidate;dihydrochloride Chemical compound Cl.Cl.COC(=N)CCSSCCC(=N)OC CQBCVFHLZAVNPF-UHFFFAOYSA-N 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 229960005485 mitobronitol Drugs 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229960003539 mitoguazone Drugs 0.000 description 1
- MXWHMTNPTTVWDM-NXOFHUPFSA-N mitoguazone Chemical compound NC(N)=N\N=C(/C)\C=N\N=C(N)N MXWHMTNPTTVWDM-NXOFHUPFSA-N 0.000 description 1
- VFKZTMPDYBFSTM-GUCUJZIJSA-N mitolactol Chemical compound BrC[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)CBr VFKZTMPDYBFSTM-GUCUJZIJSA-N 0.000 description 1
- 229950010913 mitolactol Drugs 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 229960000350 mitotane Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- YVIIHEKJCKCXOB-STYWVVQQSA-N molport-023-276-178 Chemical compound C([C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CSSC[C@H]2C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N3CCC[C@H]3C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@H](C(N[C@@H](CSSC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N2)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)=O)CC(C)C)[C@@H](C)O)C(N)=O)C1=CNC=N1 YVIIHEKJCKCXOB-STYWVVQQSA-N 0.000 description 1
- FOYWNSCCNCUEPU-UHFFFAOYSA-N mopidamol Chemical compound C12=NC(N(CCO)CCO)=NC=C2N=C(N(CCO)CCO)N=C1N1CCCCC1 FOYWNSCCNCUEPU-UHFFFAOYSA-N 0.000 description 1
- 229950010718 mopidamol Drugs 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000036438 mutation frequency Effects 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- QCQYVCMYGCHVMR-AAZUGDAUSA-N n-[(2r,3r,4s,5r)-4,5,6-trihydroxy-1-oxo-3-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexan-2-yl]acetamide Chemical compound CC(=O)N[C@@H](C=O)[C@H]([C@@H](O)[C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O QCQYVCMYGCHVMR-AAZUGDAUSA-N 0.000 description 1
- NJSMWLQOCQIOPE-OCHFTUDZSA-N n-[(e)-[10-[(e)-(4,5-dihydro-1h-imidazol-2-ylhydrazinylidene)methyl]anthracen-9-yl]methylideneamino]-4,5-dihydro-1h-imidazol-2-amine Chemical compound N1CCN=C1N\N=C\C(C1=CC=CC=C11)=C(C=CC=C2)C2=C1\C=N\NC1=NCCN1 NJSMWLQOCQIOPE-OCHFTUDZSA-N 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 229940086322 navelbine Drugs 0.000 description 1
- QZGIWPZCWHMVQL-UIYAJPBUSA-N neocarzinostatin chromophore Chemical compound O1[C@H](C)[C@H](O)[C@H](O)[C@@H](NC)[C@H]1O[C@@H]1C/2=C/C#C[C@H]3O[C@@]3([C@@H]3OC(=O)OC3)C#CC\2=C[C@H]1OC(=O)C1=C(O)C=CC2=C(C)C=C(OC)C=C12 QZGIWPZCWHMVQL-UIYAJPBUSA-N 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229960001267 nesiritide Drugs 0.000 description 1
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)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(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)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)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 description 1
- 229940071846 neulasta Drugs 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- XWXYUMMDTVBTOU-UHFFFAOYSA-N nilutamide Chemical compound O=C1C(C)(C)NC(=O)N1C1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 XWXYUMMDTVBTOU-UHFFFAOYSA-N 0.000 description 1
- 229960002653 nilutamide Drugs 0.000 description 1
- 229960001420 nimustine Drugs 0.000 description 1
- VFEDRRNHLBGPNN-UHFFFAOYSA-N nimustine Chemical compound CC1=NC=C(CNC(=O)N(CCCl)N=O)C(N)=N1 VFEDRRNHLBGPNN-UHFFFAOYSA-N 0.000 description 1
- YMVWGSQGCWCDGW-UHFFFAOYSA-N nitracrine Chemical compound C1=CC([N+]([O-])=O)=C2C(NCCCN(C)C)=C(C=CC=C3)C3=NC2=C1 YMVWGSQGCWCDGW-UHFFFAOYSA-N 0.000 description 1
- 229950008607 nitracrine Drugs 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229950009266 nogalamycin Drugs 0.000 description 1
- KGTDRFCXGRULNK-JYOBTZKQSA-N nogalamycin Chemical compound CO[C@@H]1[C@@](OC)(C)[C@@H](OC)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=C(O)C=C4[C@@]5(C)O[C@H]([C@H]([C@@H]([C@H]5O)N(C)C)O)OC4=C3C3=O)=C3C=C2[C@@H](C(=O)OC)[C@@](C)(O)C1 KGTDRFCXGRULNK-JYOBTZKQSA-N 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 108091008581 nuclear androgen receptors Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 1
- 230000003606 oligomerizing effect Effects 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- CZDBNBLGZNWKMC-MWQNXGTOSA-N olivomycin Chemical class O([C@@H]1C[C@@H](O[C@H](C)[C@@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1)O[C@H]1O[C@@H](C)[C@H](O)[C@@H](OC2O[C@@H](C)[C@H](O)[C@@H](O)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@H](O)[C@H](OC)[C@H](C)O1 CZDBNBLGZNWKMC-MWQNXGTOSA-N 0.000 description 1
- 229950011093 onapristone Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 108010000953 osteoblast cadherin Proteins 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940002988 pegasys Drugs 0.000 description 1
- 108010044644 pegfilgrastim Proteins 0.000 description 1
- 108010092853 peginterferon alfa-2a Proteins 0.000 description 1
- 108010092851 peginterferon alfa-2b Proteins 0.000 description 1
- 229940106366 pegintron Drugs 0.000 description 1
- 108700037519 pegvisomant Proteins 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- QIMGFXOHTOXMQP-GFAGFCTOSA-N peplomycin Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCN[C@@H](C)C=1C=CC=CC=1)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1NC=NC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QIMGFXOHTOXMQP-GFAGFCTOSA-N 0.000 description 1
- 229950003180 peplomycin Drugs 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 108010049224 perlecan Proteins 0.000 description 1
- 108700010839 phage proteins Proteins 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229960000952 pipobroman Drugs 0.000 description 1
- NJBFOOCLYDNZJN-UHFFFAOYSA-N pipobroman Chemical compound BrCCC(=O)N1CCN(C(=O)CCBr)CC1 NJBFOOCLYDNZJN-UHFFFAOYSA-N 0.000 description 1
- NUKCGLDCWQXYOQ-UHFFFAOYSA-N piposulfan Chemical compound CS(=O)(=O)OCCC(=O)N1CCN(C(=O)CCOS(C)(=O)=O)CC1 NUKCGLDCWQXYOQ-UHFFFAOYSA-N 0.000 description 1
- 229950001100 piposulfan Drugs 0.000 description 1
- 229960001221 pirarubicin Drugs 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 108010048507 poliovirus receptor Proteins 0.000 description 1
- 108010064470 polyaspartate Proteins 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- 108010000222 polyserine Proteins 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 108020001213 potassium channel Proteins 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 229960004694 prednimustine Drugs 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000002325 prokinetic agent Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 208000023958 prostate neoplasm Diseases 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000013636 protein dimer Substances 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- GPTFURBXHJWNHR-UHFFFAOYSA-N protopine Chemical compound C1=C2C(=O)CC3=CC=C4OCOC4=C3CN(C)CCC2=CC2=C1OCO2 GPTFURBXHJWNHR-UHFFFAOYSA-N 0.000 description 1
- WOLQREOUPKZMEX-UHFFFAOYSA-N pteroyltriglutamic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(=O)NC(CCC(=O)NC(CCC(O)=O)C(O)=O)C(O)=O)C(O)=O)C=C1 WOLQREOUPKZMEX-UHFFFAOYSA-N 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 229960002185 ranimustine Drugs 0.000 description 1
- 108010084837 rasburicase Proteins 0.000 description 1
- 229960000424 rasburicase Drugs 0.000 description 1
- BMKDZUISNHGIBY-UHFFFAOYSA-N razoxane Chemical compound C1C(=O)NC(=O)CN1C(C)CN1CC(=O)NC(=O)C1 BMKDZUISNHGIBY-UHFFFAOYSA-N 0.000 description 1
- 229960000460 razoxane Drugs 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 239000002265 redox agent Substances 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 108091035233 repetitive DNA sequence Proteins 0.000 description 1
- 102000053632 repetitive DNA sequence Human genes 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000004258 retinal degeneration Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 229950004892 rodorubicin Drugs 0.000 description 1
- VHXNKPBCCMUMSW-FQEVSTJZSA-N rubitecan Chemical compound C1=CC([N+]([O-])=O)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VHXNKPBCCMUMSW-FQEVSTJZSA-N 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 229950001403 sizofiran Drugs 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000003195 sodium channel blocking agent Substances 0.000 description 1
- MKNJJMHQBYVHRS-UHFFFAOYSA-M sodium;1-[11-(2,5-dioxopyrrol-1-yl)undecanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCCCCCCN1C(=O)C=CC1=O MKNJJMHQBYVHRS-UHFFFAOYSA-M 0.000 description 1
- LZWUCYAVMIKMIA-UHFFFAOYSA-M sodium;1-[3-[2-[(4-azido-2,3,5,6-tetrafluorobenzoyl)amino]ethyldisulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCSSCCNC(=O)C1=C(F)C(F)=C(N=[N+]=[N-])C(F)=C1F LZWUCYAVMIKMIA-UHFFFAOYSA-M 0.000 description 1
- ULARYIUTHAWJMU-UHFFFAOYSA-M sodium;1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O ULARYIUTHAWJMU-UHFFFAOYSA-M 0.000 description 1
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 1
- MIDXXTLMKGZDPV-UHFFFAOYSA-M sodium;1-[6-(2,5-dioxopyrrol-1-yl)hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O MIDXXTLMKGZDPV-UHFFFAOYSA-M 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 108700031632 somatrem Proteins 0.000 description 1
- 229960004532 somatropin Drugs 0.000 description 1
- 229940099077 somavert Drugs 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 229950006315 spirogermanium Drugs 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 125000002653 sulfanylmethyl group Chemical group [H]SC([H])([H])[*] 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)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](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=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](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)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](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 description 1
- 229960005460 teriparatide Drugs 0.000 description 1
- 229960005353 testolactone Drugs 0.000 description 1
- BPEWUONYVDABNZ-DZBHQSCQSA-N testolactone Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(OC(=O)CC4)[C@@H]4[C@@H]3CCC2=C1 BPEWUONYVDABNZ-DZBHQSCQSA-N 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 229960000874 thyrotropin Drugs 0.000 description 1
- 230000001748 thyrotropin Effects 0.000 description 1
- YFTWHEBLORWGNI-UHFFFAOYSA-N tiamiprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC(N)=NC2=C1NC=N2 YFTWHEBLORWGNI-UHFFFAOYSA-N 0.000 description 1
- 229950011457 tiamiprine Drugs 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- XFCLJVABOIYOMF-QPLCGJKRSA-N toremifene Chemical compound C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 XFCLJVABOIYOMF-QPLCGJKRSA-N 0.000 description 1
- 229960005026 toremifene Drugs 0.000 description 1
- 108010056110 toxin FS2 Proteins 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 229950001353 tretamine Drugs 0.000 description 1
- IUCJMVBFZDHPDX-UHFFFAOYSA-N tretamine Chemical compound C1CN1C1=NC(N2CC2)=NC(N2CC2)=N1 IUCJMVBFZDHPDX-UHFFFAOYSA-N 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- PXSOHRWMIRDKMP-UHFFFAOYSA-N triaziquone Chemical compound O=C1C(N2CC2)=C(N2CC2)C(=O)C=C1N1CC1 PXSOHRWMIRDKMP-UHFFFAOYSA-N 0.000 description 1
- 229960004560 triaziquone Drugs 0.000 description 1
- 229960001670 trilostane Drugs 0.000 description 1
- KVJXBPDAXMEYOA-CXANFOAXSA-N trilostane Chemical compound OC1=C(C#N)C[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@@]32O[C@@H]31 KVJXBPDAXMEYOA-CXANFOAXSA-N 0.000 description 1
- NOYPYLRCIDNJJB-UHFFFAOYSA-N trimetrexate Chemical compound COC1=C(OC)C(OC)=CC(NCC=2C(=C3C(N)=NC(N)=NC3=CC=2)C)=C1 NOYPYLRCIDNJJB-UHFFFAOYSA-N 0.000 description 1
- 229960001099 trimetrexate Drugs 0.000 description 1
- 229950000212 trioxifene Drugs 0.000 description 1
- 229960000875 trofosfamide Drugs 0.000 description 1
- UMKFEPPTGMDVMI-UHFFFAOYSA-N trofosfamide Chemical compound ClCCN(CCCl)P1(=O)OCCCN1CCCl UMKFEPPTGMDVMI-UHFFFAOYSA-N 0.000 description 1
- HDZZVAMISRMYHH-LITAXDCLSA-N tubercidin Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@@H](CO)[C@H](O)[C@H]1O HDZZVAMISRMYHH-LITAXDCLSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 108010072415 tumor necrosis factor precursor Proteins 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 229950009811 ubenimex Drugs 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 229960001055 uracil mustard Drugs 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine Chemical compound C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-CFWMRBGOSA-N 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004355 vindesine Drugs 0.000 description 1
- UGGWPQSBPIFKDZ-KOTLKJBCSA-N vindesine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(N)=O)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 UGGWPQSBPIFKDZ-KOTLKJBCSA-N 0.000 description 1
- GBABOYUKABKIAF-IELIFDKJSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-IELIFDKJSA-N 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
- 229940053867 xeloda Drugs 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 229950009268 zinostatin Drugs 0.000 description 1
- 229960000641 zorubicin Drugs 0.000 description 1
- FBTUMDXHSRTGRV-ALTNURHMSA-N zorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(\C)=N\NC(=O)C=1C=CC=CC=1)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 FBTUMDXHSRTGRV-ALTNURHMSA-N 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
- 108091058549 μ-conotoxin Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
-
- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
- C12N15/1044—Preparation or screening of libraries displayed on scaffold 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
Definitions
- polymers exert their effect by increasing the hydrodynamic radius (also called Stokes' radius) of the modified protein relative to the unmodified protein, which reduces the rate of clearance by kidney filtration (Yang, K., et al. (2003) Protein Eng, 16: 761-70).
- polymer attachment can reduce interaction of the modified protein with other proteins, cells, or surfaces.
- polymer attachment can reduce interactions between the modified protein and antibodies and other components of the immune system thus reducing the formation of a host immune response to the modified protein.
- protein modification by PEGylation i.e. by attaching linear or branched polymers of polyethylene glycol.
- polymers can be attached to proteins.
- hydrophilic polymers that have flexible conformations and are well hydrated in aqueous solutions.
- a frequently used polymer is polyethylene glycol (PEG).
- PEG polyethylene glycol
- the attached polymers tend to have limited interactions with the protein they have been attached to and thus the polymer-modified protein retains its relevant functions.
- the chemical conjugation of polymers to proteins requires complex multi-step processes.
- the protein component needs to be produced and purified prior to the chemical conjugation step.
- the conjugation step can result in the formation of product mixtures that need to be separated leading to significant product loss.
- such mixtures can be used as the final pharmaceutical product.
- Some examples are currently marketed PEGylated Interferon-alpha products that are used as mixtures (Wang, B. L., et al. (1998) J Submicrosc Cytol Pathol, 30: 503-9; Dhalluin, C., et al. (2005) Bioconjug Chem, 16: 504-17).
- Such mixtures are difficult to manufacture and characterize and they contain isomers with reduced or no therapeutic activity.
- cysteines for PEGylation can interfere with protein production and/or purification.
- the specific introduction of non-natural amino acids requires specific host organisms for protein production.
- a further limitation of PEGylation is that PEG is typically manufactured as a mixture of polymers with similar but not uniform length. The same limitations are inherent in many other chemical polymers.
- compositions and methods that would allow one to combine multiple polymer modules and multiple protein modules into defined multidomain products.
- the present invention provides an unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.
- URP unstructured recombinant polymer
- the present invention provides an unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP has an in vitro serum degradation half-life greater than about 24 hours, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.
- the subject URP can comprises a non-natural amino acid sequence.
- the URP is selected for incorporation into a heterologous protein, and wherein upon incorporation the URP into a heterologous protein, said heterologous protein exhibits a longer serum secretion half-life and/or higher solubility as compared to the corresponding protein that is deficient in said URP.
- the half-life can be extended by two folds, three folds, five folds, ten folds or more.
- incorporation of the URP into a heterologous protein results in at least a 2-fold, 3-fold, 4-fold, 5-fold or more increase in apparent molecular weight of the protein as approximated by size exclusion chromatography.
- the URPs has a Tepitope score less than ⁇ 3.5 (e.g., ⁇ 4 or less, ⁇ 5 or less).
- the URPs can contain predominantly hydrophilic residues.
- at least 50% of the amino acids of the URP are devoid of secondary structure as determined by Chou-Fasman algorithm.
- the glycine residues contained in the URP may constitute at least about 50% of the total amino acids of the URP.
- any one type of the amino acids alone selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) contained in the URP constitutes more than about 20%, 30%, 40%, 50%, 60% or more of the total amino acids of the URP.
- the URP comprises more than about 100, 150, 200 or more contiguous amino acids.
- the present invention also provides a protein comprising one or more of the subject URPs, wherein the subject URPs are heterologous with respect to the protein.
- the total length of URPs in aggregation can exceed about 40, 50, 60, 100, 150, 200, or more amino acids.
- the protein can comprise one or more functional modules selected from the group consisting of effector module, binding module, N-terminal module, C-terminal module, and any combinations thereof.
- the subject protein comprises a plurality of binding modules, wherein the individual binding modules exhibit binding specificities to the same or different targets.
- the binding module may comprise a disulfide-containing scaffold formed by intra-scaffold pairing of cysteines.
- the binding module may bind to a target molecule target is selected from the group consisting of cell surface protein, secreted protein, cytosolic protein, and nuclear protein.
- the target can be an ion channel and/or GPCR.
- the effector module can be a toxin.
- the subject URP-containing protein typically an extended serum secretion half-life by at least 2, 3, 4, 5, 10 or more folds as compared to a corresponding protein that is deficient in said URP.
- the present invention provides a non-naturally occurring protein comprising at least 3 repeating units of amino acid sequences, each of the repeating unit comprising at least 6 amino acids, wherein the majority of segments comprising about 6 to about 15 contiguous amino acids of the at least 3 repeating units are present in one or more native human proteins.
- the majority of the segments, or each segment comprising about 9 to about 15 contiguous amino acids within the repeating units are present in one or more native human proteins.
- the segments can comprise about 9 to about 15 amino acids.
- the three repeating units may share substantial sequence homology, e.g., share sequence identify of greater than about 50%, 60%, 70%, 80%, 90% or 100% when aligned.
- non-natural protein may also comprise one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.
- the non-natural protein may comprise individual repeating unit having the subject unstructured recombinant polymer (URP).
- the present invention also provides recombinant polynucleotides comprising coding sequences that encode the subject URPs, URP-containing proteins, microproteins and toxins. Also provided in the present invention are vectors containing the subject polynucleotides, host cells harboring the vectors, genetic packages displaying the subject URPs, URP-containing proteins, toxins and any other proteinaceous entities disclosed herein. Further provided are selectable library of expression vectors of the present invention.
- the present invention also provides method of producing a protein comprising an unstructured recombinant polymer (URP).
- the method involves (i) providing a host cell comprising a recombinant polynucleotide encoding the protein, said protein comprising one or more URP, said URP comprising at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm; and (ii) culturing said host cell in a suitable culture medium under conditions to effect expression of said protein from said polynucleotide.
- the present invention also provides a method of increasing-serum secretion half-life of a protein, comprising: fusing said protein with one or more unstructured recombinant polymers (URPs), wherein the URP comprises at least about 40 contiguous amino acids, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm; and wherein said URP is substantially incapable of non-specific binding to a serum protein.
- URPs unstructured recombinant polymers
- Also provided in the present invention is a method of detecting the presence or absence of a specific interaction between a target and an exogenous protein that is displayed on a genetic package, wherein said protein comprises one or more unstructured recombinant polymer (URP), the method comprising: (a) providing a genetic package displaying a protein that comprises one or more unstructured recombinant polymers (URPs); (b) contacting the genetic package with the target under conditions suitable to produce a stable protein-target complex; and (c) detecting the formation of the stable protein-target complex on the genetic package, thereby detecting the presence of a specific interaction.
- the method may further comprises obtaining a nucleotide sequence from the genetic package that encodes the exogenous protein.
- the presence or absence of a specific interaction is between the URP and a target comprising a serum protein.
- the presence or absence of a specific interaction is between the URP and a target comprising a serum protease.
- microprotein retains binding capability to its native target.
- the microprotein exhibits binding capability towards at least one family of ion channel selected from the group consisting of a sodium, a potassium, a calcium, an acetylcholine, and a chlorine channel.
- the microprotein is an ion-channel-binding microprotein, and is modified such that (a) the microprotein binds to a different family of channel as compared to the corresponding unmodified microprotein; (b) the microprotein binds to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein; (c) the microprotein binds to a different species of the same subfamily of channel as compared to the corresponding unmodified microprotein; (d) the microprotein binds to a different site on the same channel as compared to the corresponding unmodified microprotein; and/or (e) the microprotein binds to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein.
- the microprotein is a toxin.
- the present invention also provides a library of genetic packages displaying the subject microproteins and/or toxins. Where desired, the genetic package displays a proteinaceous toxin that retains in part or in whole its toxicity spectrum.
- the toxin can be derived from a single toxin protein, or derived from a family of toxins.
- the present invention also provides a library of genetic packages wherein the library displays a family of toxins, wherein the family retains in part or in whole its native toxicity spectrum.
- the present invention further provides a protein comprising a plurality of ion-channel binding domains, wherein individual domains are microprotein domains that have been modified such that (a) the microprotein domains bind to a different family of channel as compared to the corresponding unmodified microprotein domains; (b) the microprotein domains bind to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein domains; (c) the microprotein domains bind to a different species of the same subfamily as compared to the corresponding unmodified microprotein domains; (d) the microprotein domains bind to a different site on the same channel as compared to the corresponding unmodified microprotein domains; (e) the microprotein domains bind to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein domains; and/or (f) the microprotein domains bind to the same site of the same channel and yield the same biological effect as compared to the corresponding unmodified
- Also embodied in the invention is a method of obtaining a microprotein with desired property, comprising: (a) providing a subject library; and (b) screening the selectable library to obtain at least one phage displaying a microprotein with the desired property.
- Polynucleotides, vectors, genetic packages, host cells for use in any one of the disclosed methods are also provided.
- FIG. 1 shows the modular components of an MURP. Binding modules, effector modules, and multimerization modules are depicted as circles. URP modules, N-terminal, and C-terminal modules are shown as rectangles.
- FIG. 2 shows examples of modular architectures of MURPs.
- Binding modules (BM) in one MURP can have identical or differing target specificities.
- FIG. 3 shows that a repeat protein that is based on a human sequence can contain novel amino acid sequences, which can contain T cell epitopes. These novel sequences are formed at the junction between neighboring repeat units.
- FIG. 4 illustrates the design of a URP sequence that is a repeat protein based on three human donor sequences D1, D2, and D3.
- the repeating unit of this URP was chosen such that even 9-mer sequences that span the junction between neighboring units can be found in at least one of the human donor sequences.
- FIG. 5 Example of a URP sequences that is a repeat protein based on the sequences of three human proteins. The lower portion of the figure illustrates that all 9-mer subsequences in the URP occur in at least one of the human donor proteins.
- FIG. 6 Example based URP sequence based on the human POU domain residues 146-182.
- FIG. 7 shows the advantage of separating modules with information rich sequences by inserting URP modules between such sequences.
- the left side of the figure shows that the direct fusion of modules A and B leads to novel sequences in the junction region. These junction sequences can be epitopes.
- the right half of the figure shows that the insertion of a URP module between module A and B prevents the formation of such junction sequences that contain partial sequences from modules A and B. Instead, the termini of modules A and B yield junction sequences that contain URP sequences and thus are predicted to have low immunogenicity.
- FIG. 8 shows drug delivery constructs that are based on URPs.
- the drug molecules depicted as hexagons are chemically conjugated to the MURP.
- FIG. 9 shows and MURP containing a protease-sensitive site.
- the URP module is designed such that it blocks the effector module from its function. Protease cleavage removes a portion of the URP module and results in increased activity of the effector function.
- FIG. 10 shows how an URP module can act as a linker between a binding module and an effector module.
- the binding module can bind to a target and as a consequence it increases the local concentration of the effector module in the proximity of the target.
- FIG. 11 Shows a process to construct genes encoding URP sequences from libraries of short URP modules.
- the URP module library can be inserted into a stuffer vector that contains green fluorescent protein (GFP) as a reporter to facilitate the identification of URP sequences with high expression.
- GFP green fluorescent protein
- FIG. 12 shows MURPs that contain multiple binding modules for death receptors. Death receptors are triggered by trimerization and thus MURPs containing at least three binding elements for one death receptor particularly potent in inducing cell death. The lower portion of the figure illustrates that one can increase the specificity of the MURP for diseased tissue by adding one or more binding modules with specificity for tumor tissue.
- FIG. 13 shows a MURP that comprises four binding modules (rectangles) with specificity for a tumor antigen with an effector module like interleukin 2.
- FIG. 14 shows the flow chart for the construction of URP modules with 288 residues.
- the URP modules were constructed as fusion proteins with GFP. Libraries of URP modules with 36 amino acids were constructed first followed by iterative dimerization to yield URP modules with 288 amino acids (rPEG_H288 and rPEG_J288).
- FIG. 15 Amino acid and nucleotide sequence of a URP module with 288 amino acids (rPEG_J288).
- FIG. 16 Amino acid and nucleotide sequence of a URP module with 288 amino acids (rPEG_H288).
- FIG. 17 Amino acid sequence of a serine-rich sequence region of the human protein dentin sialophosphoprotein.
- FIG. 18 shows a depot derivative of a MURP.
- the protein contains two cysteine residues that can form a weak SS bridge.
- the protein can be manufactured with the SS bridge intact. It can be formulated and injected into patients in reduced form. After injection it will be oxidized in proximity to the injection site and as a result in can form a high molecular weight polymer with very limited diffusivity.
- the active MURP can slowly leach from the injection site by limited proteolysis or limited reduction of the cross linking SS bond.
- FIG. 19 shows a depot form of a MURP.
- the MURP has very limited diffusivity at the injection site and can be liberated from the injection site by limited proteolysis.
- FIG. 20 shows a depot form of a MURP that contains a histidine-rich sequence.
- the MURP can be formulated and injected in combination with insoluble beads that contain immobilized nickel.
- the MURP binds to the nickel beads at the injection site and is released slowly into the circulation.
- FIG. 21 shows MURPs that contain multimerization modules.
- the upper part of the figure shows an MURP that contains one dimerization sequence. As a result it forms a dimer which effectively doubles its molecular weight.
- the center of the figure shows three MURP designs that comprise two multimerization sequences. Such MURPs can form multimers with very high effective molecular weight.
- FIG. 22 Shows a variety of MURPs that are designed to block or modulate ion channel function. Circles indicate binding modules with specificity for ion channels. These binding modules can be derived or identical to natural toxins with affinity for ion channel receptors. The figure illustrates that other binding domains can be added on either side of the ion channel-specific binding modules thus conferring the MURPs increased efficacy or specificity for a particular cell type.
- FIG. 23 shows several MURP designs for increased half-life. Increased effective molecular weight can be achieved by increasing chain length (A), chemical multimerization (B), adding multiple copies of binding modules into a molecule separated by non-binding sites (C), construction of chemical multimers similar to C (D, E), including multimerization sequences (F).
- FIG. 24 shows MURPs that can be formed by chemical conjugation of binding modules to a recombinant URP sequence.
- the URP sequence is designed to contain multiple lysine residues (K) as conjugation sites.
- FIG. 25 shows the design of a library of 2SS binding modules.
- the sequences contain a constant 1SS sequence in the center which is flanked by random sequences that contain cysteine residues in varying distance from the 1SS core.
- FIG. 26 shows the design of a library of 2SS binding modules.
- the sequences contain a constant 1SS sequence in the center which is flanked by random sequences that contain cysteine residues in varying distance from the 1SS core.
- FIG. 27 shows the design of a library of dimers of 1SS binding modules. Initially, a collection of 1SS binding modules is amplified by two PCR reactions. The resulting PCR products are combined and dimers are generated in a subsequent PCR step.
- FIG. 28 show the Western analysis of a fusion protein containing the 288 amino acid URP sequence rPEG_J288 after incubation of up to 3 days in 50% mouse serum.
- FIG. 29 shows results of a binding assay testing for pre-existing antibodies against a URP sequence of 288 amino acids.
- FIG. 30 shows the binding of MURPs containing one (Monomer), two (Dimer), four (Tetramer), or zero (rPEG36) binding modules with specificity for VEGF which was coated to microtiter plates.
- FIG. 31 shows the amino acid sequence of an MURP with specificity for EpCAM.
- the sequence contains four binding modules with affinity for EpCAM (underlined).
- the sequence contains an N-terminal Flag sequence which contains the only two lysine residues of the entire sequence.
- FIG. 32 shows the design of 1SS addition libraries. Random 1SS modules can be added to the N- or C-terminus of a pre-selected binding module or simultaneously to both sides.
- FIG. 33 shows the alignment of three finger toxin-related sequences. The figure also shows a 3D structure that was solved by NMR.
- FIG. 34 shows the design of a three-finger toxin-based library. Residues designated X were randomized. The codon choice for each random position is indicated.
- FIG. 35 shows the alignment of plexin-related sequences.
- FIG. 36 shows the design of a plexin-based library. Residues designated X were randomized. The codon choice for each random position is indicated.
- FIG. 37 Sequences of plexin-related binding modules with specificity for DR4, ErbB2, and HGFR.
- FIG. 38 shows a binding assay for microprotein-based binding domains with specificity for VEGF.
- FIG. 39 shows sequences of 2SS and 3SS binding modules that were isolated from buildup libraries with specificity for VEGF.
- the upper part of the protein shows PAGE gel analysis of the proteins purified by heat-lysis.
- FIG. 40 shows cloning steps to construct the URP sequence rPEG_J72.
- FIG. 41 shows the construction of a library of URP modules with 36 amino acids called rPEG_J36.
- the region encoding rPEG_J36 was assembled by ligating three shorter segments encoding rPEG_J12 and a stopper module.
- FIG. 42 shows the nucleotide sequence and translation of the stuffer vector pCW0051.
- the stuffer region is flanked by BsaI and BbsI sites and contains multiple stop codons.
- FIG. 43 shows a PAGE gel of the purification of the URP rPEG_J288 fused to GFP.
- Lane 2 shows the cell lysate; lane 3: product purified by IMAC; lane 4: product purified by anti-Flag.
- FIG. 44 Amino acid sequence of fusion proteins between rPEG_J288 and human effector domains interferon alpha, G-CSF, and human growth hormone.
- FIG. 45 shows the Western analysis of expression of fusion proteins between rPEG_J288 and human growth hormone (lanes 1 and 2), interferon alpha (lanes 3 and 4), and GFP (lanes 5 and 6). Both soluble and insoluble material was analyzed for each protein.
- FIG. 46 shows the design of MURPs based on the toxin OSK1. The figure shows that URP sequences and/or binding modules can be added to either side of OSK1
- FIG. 47 depicts exemplary product formats comprising the subject URPs.
- a cell includes a plurality of cells, including mixtures thereof.
- polypeptide refers to polymers of amino acids of any length.
- the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
- the terms also encompass an amino acid polymer that has been modified, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
- amino acid refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
- a “repetitive sequence” refers to an amino acid sequence that can be described as an oligomer of repeating peptide sequences, forming direct repeats, or inverted repeats or alternating repeats of multiple sequence motifs. These repeating oligomer sequences can be identical or homologous to each other, but there can also be multiple repeated motifs. Repetitive sequences are characterized by a very low information content. A repetitive sequence is not a required feature of a URP and in some cases a non-repetitive sequence will in fact be preferred.
- Amino acids can be characterized based on their hydrophobicity.
- a number of scales have been developed. An example is a scale developed by Levitt, M et al. (see Levitt, M (1976) J Mol Biol 104, 59, #3233, which is listed in Hopp, T P, et al. (1981) Proc Natl Acad Sci USA 78, 3824, #3232).
- “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine.
- hydrophobic amino acids are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
- denatured conformation describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperatures. Peptides in denatured conformation have characteristic CD spectra and they are generally characterized by a lack of long range interactions as determined by e.g., NMR. Denatured conformation and unfolded conformation will be used synonymously.
- URP sequences and “unstructured recombinant polymer” (URP) are used herein interchanageably.
- the terms refer to amino acid sequences that share commonality with denatured peptide sequences, e.g., exhibiting a typical behavior like denatured peptide sequences, under physiological conditions, as detailed herein.
- URP sequences lack a defined tertiary structure and they have limited or no secondary structure as detected by, e.g., Chou-Fasman algorithm.
- cell surface proteins refers to the plasma membrane components of a cell. It encompasses integral and peripheral membrane proteins, glycoproteins, polysaccharides and lipids that constitute the plasma membrane.
- An integral membrane protein is a transmembrane protein that extends across the lipid bilayer of the plasma membrane of a cell.
- a typical integral membrane protein consists of at least one membrane spanning segment that generally comprises hydrophobic amino acid residues. Peripheral membrane proteins do not extend into the hydrophobic interior of the lipid bilayer and they are bound to the membrane surface via covalent or noncovalent interaction directly or indirectly with other membrane components.
- membrane cytosolic
- nuclear cytoplasmic
- secreted as applied to cellular proteins specify the extracellular and/or subcellular location in which the cellular protein is mostly, predominantly, or preferentially localized.
- Cell surface receptors represent a subset of membrane proteins, capable of binding to their respective ligands.
- Cell surface receptors are molecules anchored on or inserted into the cell plasma membrane. They constitute a large family of proteins, glycoproteins, polysaccharides and lipids, which serve not only as structural constituents of the plasma membrane, but also as regulatory elements governing a variety of biological functions.
- module refers to a portion of a protein that is physically or functionally distinguished from other portions of the protein or peptide.
- a module can comprise one or more domains.
- a module or domain can be a single, stable three-dimensional structure, regardless of size.
- the tertiary structure of a typical domain is stable in solution and remains the same whether such a member is isolated or covalently fused to other domains.
- a domain generally has a particular tertiary structure formed by the spatial relationships of secondary structure elements, such as beta-sheets, alpha helices, and unstructured loops.
- disulfide bridges are generally the primary elements that determine tertiary structure.
- domains are modules that can confer a specific functional activity, such as avidity (multiple binding sites to the same target), multi-specificity (binding sites for different targets), halflife (using a domain, cyclic peptide or linear peptide) which binds to a serum protein like human serum albumin (HSA) or to IgG (hIgG1, 2, 3 or 4) or to red blood cells.
- Functionally-defined domains have a distinct biological function(s).
- the ligand-binding domain of a receptor for example, is that domain that binds ligand.
- An antigen-binding domain refers to the part of an antigen-binding unit or an antibody that binds to the antigen.
- Functionally-defined domains need not be encoded by contiguous amino acid sequences.
- Functionally-defined domains may contain one or more physically-defined domain.
- Receptors for example, are generally divided into the extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain.
- a “membrane anchorage domain” refers to the portion of a protein that mediates membrane association. Generally, the membrane anchorage domain is composed of hydrophobic amino acid residues. Alternatively, the membrane anchorage domain may contain modified amino acids, e.g. amino acids that are attached to a fatty acid chain, which in turn anchors the protein to a membrane.
- Non-naturally occurring as applied to a protein means that the protein contains at least one amino acid that is different from the corresponding wildtype or native protein.
- Non-natural sequences can be determined by performing BLAST search using, e.g., the lowest smallest sum probability where the comparison window is the length of the sequence of interest (the queried) and when compared to the non-redundant (“nr”) database of Genbank using BLAST 2.0.
- the BLAST 2.0 algorithm which is described in Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
- a “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors.
- Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
- a host cell includes cells transfected in vivo with a vector of this invention.
- isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. As is apparent to those of skill in the art, a non-naturally occurring the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.
- a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
- Linked and “fused” or “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements or components, by whatever means including chemical conjugation or recombinant means.
- An “in-frame fusion” refers to the joining of two or more open reading frames (OFRs) to form a continuous longer OFR, in a manner that maintains the correct reading frame of the original OFRs.
- the resulting recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original OFRs (which segments are not normally so joined in nature.)
- a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
- a “partial sequence” is a linear sequence of part of a polypeptide which is known to comprise additional residues in one or both directions.
- Heterologous means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence.
- heterologous as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
- polynucleotides refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
- polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
- a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
- modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
- the sequence of nucleotides may be interrupted by non-nucleotide components.
- a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
- Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
- gene or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
- a gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof.
- a “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
- a “vector” is a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
- the term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.
- An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s).
- An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
- the “target” as used in the context of MURPs is a biochemical molecule or structure to which the Binding Module or the URP-linked Binding Module can bind and where the binding event results in a desired biological activity.
- the target can be a protein ligand or receptor that is inhibited, activated or otherwise acted upon by the t protein.
- targets are hormones, cytokines, antibodies or antibody fragments, cell surface receptors, kinases, growth factors and other biochemical structures with biological activity.
- a “functional module” can be any non-URP in a protein product.
- a functional module can be a binding module (BM), an effector module (EM), a multimerization module (MM), a C-terminal module (CM), or an N-terminal module (NM).
- BM binding module
- EM effector module
- MM multimerization module
- CM C-terminal module
- NM N-terminal module
- functional modules are characterized by a high information content of their amino acid sequence, i.e they contain many different amino acids and many of these amino acids are important for the function of a functional module.
- a functional module typically has secondary and tertiary structure, may be a folded protein domain and may contain 1, 2, 3, 4, 5 or more disulfide bonds.
- microproteins refers to a classification in the SCOP database. Microproteins are usually the smallest proteins with a fixed structure and typically but not exclusively have as few as 15 amino acids with two disulfides or up to 200 amino acids with more than ten disulfides. A microprotein may contain one or more microprotein domains. Some microprotein domains or domain families can have multiple more-or-less stable and multiple more or less similar structures which are conferred by different disulfide bonding patterns, so the term stable is used in a relative way to differentiate microproteins from peptides and non-microprotein domains. Most microprotein toxins are composed of a single domain, but the cell-surface receptor microproteins often have multiple domains.
- Microproteins can be so small because their folding is stabilized either by disulfide bonds and/or by ions such as Calcium, Magnesium, Manganese, Copper, Zinc, Iron or a variety of other multivalent ions, instead of being stabilized by the typical hydrophobic core.
- ions such as Calcium, Magnesium, Manganese, Copper, Zinc, Iron or a variety of other multivalent ions, instead of being stabilized by the typical hydrophobic core.
- scaffold refers to the minimal polypeptide ‘framework’ or ‘sequence motif’ that is used as the conserved, common sequence in the construction of protein libraries.
- a scaffold is typically defined by the conserved residues that are observed in an alignment of a family of sequence-related proteins. Fixed residues may be required for folding or structure, especially if the functions of the aligned proteins are different.
- a full description of a microprotein scaffold may include the number, position or spacing and bonding pattern of the cysteines, as well as position and identity of any fixed residues in the loops, including binding sites for ions such as Calcium.
- the “fold” of a microprotein is largely defined by the linkage pattern of the disulfide bonds (i.e., 1-4, 2-6, 3-5). This pattern is a topological constant and is generally not amenable to conversion into another pattern without unlinking and relinking the disulfides such as by reduction and oxidation (redox agents).
- redox agents reduction and oxidation
- natural proteins with related sequences adopt the same disulfide bonding patterns.
- the major determinants are the cysteine distance pattern (CDP) and some fixed non-cys residues, as well as a metal-binding site, if present.
- the folding of proteins is also influenced by the surrounding sequences (ie pro-peptides) and in some cases by chemical derivatization (ie gamma-carboxylation) of residues that allow the protein to bind divalent metal ions (ie Ca++) which assists their folding.
- the surrounding sequences ie pro-peptides
- chemical derivatization ie gamma-carboxylation
- proteins with the same bonding pattern may still comprise multiple folds, based on differences in the length and composition of the loops that are large enough to give the protein a rather different structure.
- An example are the conotoxin, cyclotoxin and anato domain families, which have the same DBP but a very different CDP and are considered to be different folds.
- Determinants of a protein fold are any attributes that greatly alter structure relative to a different fold, such as the number and bonding pattern of the cysteines, the spacing of the cysteines, differences in the sequence motifs of the inter-cysteine loops (especially fixed loop residues which are likely to be needed for folding, or in the location or composition of the calcium (or other metal or co-factor) binding site.
- DBP disulfide bonding pattern
- cysteines which are numbered 1-n from the N-terminus to the C-terminus of the protein.
- Disulfide bonding patterns are topologically constant, meaning they can only be changed by unlinking one or more disulfides such as using redox conditions.
- the possible 2-, 3-, and 4-disulfide bonding patterns are listed below in paragraphs 0048-0075.
- CDP cyste distance pattern
- Serum degradation resistance Proteins can be eliminated by degradation in the blood, which typically involves proteases in the serum or plasma.
- the serum degradation resistance is measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (ie 0.25, 0.5, 1, 2, 4, 8, 16 days) at 37 C.
- the samples for these timepoints are then run on a western assay and the protein is detected with an antibody.
- the antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred.
- the timepoint where 50% of the protein is degraded, as judged by western is the serum degradation halflife of the protein.
- Serum protein binding While the MURP typically has a number of modules that bind to cell-surface targets and/or serum proteins, it is desirable that the URP substantially lack unintended activities.
- the URP should be designed to minimize avoid interaction with (binding to) serum proteins, including antibodies.
- Different URP designs can be screened for serum protein binding by ELISA, immobilizing the serum proteins and then adding the URP, incubating, washing and then detecting the amount of bound URP.
- One approach is to detect the URP using an antibody that recognizes a tag that has been added to the URP.
- a different approach is to immobilize the URP (such as via a fusion to GFP) and come in with human serum, incubating, washing, and then detecting the amount of human antibodies that remain bound to the URP using secondary antibodies like goat anti-human IgG.
- URPs we have designed our URPs to show very low levels of binding to serum proteins.
- binding to serum proteins or serum-exposed proteins is desired, for example because it can further extend the secretion halflife.
- the MURP can be given binding modules that contain peptides that have been designed to bind to serum proteins or serum-exposed proteins such as HAS or IgG.
- URPs unstructured recombinant polymers
- the subject URPs are particularly useful for generating recombinant proteins of therapeutic and/or diagnostic value.
- the subject URPs exhibit one or more following features.
- the subject URPs comprise amino acid sequences that typically share commonality with denatured peptide sequences under physiological conditions.
- URP sequences typically behave like denatured peptide sequences under physiological conditions.
- URP sequences lack well defined secondary and tertiary structures under physiological conditions.
- a variety of methods have been established in the art to ascertain the second and tertiary structures of a given polypeptide.
- the secondary structure of a polypeptide can be determined by CD spectroscopy in the “far-UV” spectral region (190-250 nm). Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectra.
- Secondary structure can also be ascertained via certain computer programs or algorithms such as the Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45). For a given URP sequence, the algorithm can predict whether there exists some or no secondary structure at all. In general, URP sequences will have spectra that resemble denatured sequences due to their low degree of secondary and tertiary structure. Where desired, URP sequences can be designed to have predominantly denatured conformations under physiological conditions. URP sequences typically have a high degree of conformational flexibility under physiological conditions and they tend to have large hydrodynamic radii (Stokes' radius) compared to globularproteins of similar molecular weight.
- physiological conditions refer to a set of conditions including temperature, salt concentration, pH that mimic those conditions of a living subject.
- a host of physiologically relevant conditions for use in in vitro assays have been established.
- a physiological buffer contains a physiological concentration of salt and at adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
- a variety of physiological buffers is listed in Sambrook et al. (1989) supra and hence is not detailed herein.
- Physiologically relevant temperature ranges from about 25° C. to about 38° C., and preferably from about 30° C. to about 37° C.
- the subject URPs can be sequences with low immunogenicity.
- Low immunogenicity can be a direct result of the conformational flexibility of URP sequences.
- Many antibodies recognize so-called conformational epitopes in protein antigens. Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The precise folding of the protein brings these sequences into a well-defined special configuration that can be recognized by antibodies.
- Preferred URPs are designed to avoid formation of conformational epitopes.
- URP sequences having a low tendency to adapt compactly folded conformations in aqueous solution.
- low immunogenicity can be achieved by choosing sequences that resist antigen processing in antigen presenting cells, choosing sequences that do not bind MHC well and/or by choosing sequences that are derived from human sequences.
- the subject URPs can be sequences with a high degree of protease resistance.
- Protease resistance can also be a result of the conformational flexibility of URP sequences.
- Protease resistance can be designed by avoiding known protease recognition sites.
- protease resistant sequences can be selected by phage display or related techniques from random or semi-random sequence libraries.
- serum protease cleavage sites can be built into an URP.
- URP sequences with high stability e.g., long serum half-life, less prone to cleavage by proteases present in bodily fluid
- the subject URP can also be characterized by the effect in that wherein upon incorporation of it into a protein, the protein exhibits a longer serum half-life and/or higher solubility as compared to the corresponding protein that is deficient in the URP.
- Methods of ascertaining serum half-life are known in the art (see e.g., Alvarez, P., et al. (2004) J Biol Chem, 279: 3375-81).
- One can readily determine whether the resulting protein has a longer serum half-life as compared to the unmodified protein by praciting any methods available in the art or exemplified herein.
- the subject URP can be of any length necessary to effect (a) extension of serum half-life of a protein comprising the URP; (b) an increase in solubility of the resulting protein; (c) an increased resistance to protease; and/or (d) a reduced immunogenicity of the resulting protein that comprises the URP.
- the subject URP has about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or more contiguous amino acids.
- the URP can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs.
- Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 40 amino acids.
- the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- URPs may have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 or even 13.0.
- pI isoelectric point
- URP sequences are rich in hydrophilic amino acids and contain a low percentage of hydrophobic or aromatic amino acids.
- Suitable hydrophilic residues include but are not limited to glycine, serine, aspartate, glutamate, lysine, arginine, and threonine.
- Hydrophobic residues that are less favored in construction of URPs include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine.
- URP sequences can be rich in glycine but URP sequences can also be rich in the amino acids glutamate, aspartate, serine, threonine, alanine or proline.
- the predominant amino acid may be G, E, D, S, T, A or P.
- the inclusion of proline residues tends to reduce sensitivity to proteolytic degradation.
- URP sequences typically increase URPs' solubility in water and aqueous media under physiological conditions.
- URP sequences have a low tendency to form aggregates in aqueous formulations and the fusion of URP sequences to other proteins or peptides tends to enhance their solubility and reduce their tendency to form aggregates, which is a separate mechanism to reduce immunogenicity.
- URP sequences can be designed to avoid certain amino acids that confer undesirable properties to the protein. For instance, one can design URP sequences to contain few or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation).
- the subject URP comprises a glycine rich sequence (GRS).
- GRS glycine rich sequence
- glycine can be present predominantly such that it is the most prevalent residues present in the sequence of interest.
- URP sequences can be designed such that glycine residues constitute at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the total amino acids.
- URPs can also contain 100% glycines.
- the URPs contain at least 30% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 20%.
- the URPs contain at least 40% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 10%. In still yet another example, the URPs contain at least about 50% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 5%.
- the length of GRS can vary between about 5 amino acids and 200 amino acids or more.
- the length of a single, contiguous GRS can contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 240, 280, 320 or 400 or more amino acids.
- GRS may comprise glycine residues at both ends.
- GRS can also have a significant content of other amino acids, for example Ser, Thr, Ala, or Pro.
- GRS can contain a significant fraction of negatively charged amino acids including but not limited to Asp and Glu.
- GRS can contain a significant fraction of positively charged amino acids including but not limited to Arg or Lys.
- URPs can be designed to contain only a single type of amino acid (i.e., Gly or Glu), sometimes only a few types of amino acid, e.g., two to five types of amino acids (e.g., selected from G, E, D, S, T, A and P), in contrast to typical proteins and typical linkers which generally are composed of most of the twenty types of amino acids.
- URPs may contain negatively charged residue's (Asp, Glu) in 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of the amino acids positions.
- the subject GRS-containing URP has about 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids.
- the URP can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs. Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 30 amino acids.
- the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, or more amino acids.
- the GRS-containing URPs are of particular interest due to, in part, the increased conformational freedom of glycine-containing peptides.
- Denatured peptides in solution have a high degree of conformational freedom. Most of that conformational freedom is lost upon binding of said peptides to a target like a receptor, an antibody, or a protease. This loss of entropy needs to be offset by the energy of interaction between the peptide and its target.
- the degree of conformational freedom of a denatured peptide is dependent on its amino acid sequences. Peptides containing many amino acids with small side chains tend to have more conformational freedom than peptides that are composed of amino acids with larger side chains.
- Peptides containing the amino acid glycine have particularly large degrees of freedom. It has been estimated that glycine-containing peptide bonds have about 3.4 times more entropy in solution as compared to corresponding alanine-containing sequences (D'Aquino, J. A., et al. (1996) Proteins, 25: 143-56). This factor increases with the number of glycine residues in a sequence. As a result, such peptides tend to lose more entropy upon binding to targets, which reduces their overall ability to interact with other proteins as well as their ability to adopt defined three-dimensional structures.
- GRS-containing URPs are sequences from human proteins or sequences that share substantial homology to the corresponding glycine rich sequences in the reference human proteins.
- G small conductance calcium-activated potassium channel protein 2 isoform a; apamin-sensitive small-conductance Ca2+-activated potassium channel [ Homo sapiens ] G T GGGG ST GGGGGGGG S G H G SRY (sex determining region Y)-box 1; SRY-related HMG-box gene 1 [ Homo sapiens ] G PA G A GGGGGGGGGGGGG transcription factor 20 isoform 2; stromelysin-1 platelet-derived growth factor-responsive element binding protein; stromelysin 1 PDGF-responsive element-binding protein; SPRE-binding protein; nuclear factor SPBP [ Homo sapiens ] GG T GG SS G SS G S G S GGG RR G transcription factor 20 isoform 1; stromelysin-1 platelet-derived growth factor-derived growth factor-derived growth factor-responsive element binding protein; stromelysin 1 PDGF-responsive element-binding protein; SPRE-binding protein; nuclear factor SPBP [ Homo sap
- NGR Non-Glycine Residues
- sequences of non-glycine residues in these GRS can be selected to optimize the properties of URPs and hence the proteins that contain the desired URPs. For instance, one can optimize the sequences of URPs to enhance the selectivity of the resulting protein for a particular tissue, specific cell type or cell lineage. For example, one can incorporate protein sequences that are not ubiquitously expressed, but rather are differentially expressed in one or more of the body tissues including heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, and selected tissues that are affected by diseases such as infectious diseases, autoimmune disease, renal, neronal, cardiac disorders and cancers.
- tissue-specific, cell-type specific, subcellular location specific sequences are known and available from numerous protein databases.
- Such selective URP sequences can be obtained by generating libraries of random or semi-random URP sequences, injecting them into animals or patients, and determining sequences with the desired tissue selectivity in tissue samples. Sequence determination can be performed by mass spectrometry. Using similar methods one can select URP sequences that facilitate oral, buccal, intestinal, nasal, thecal, peritoneal, pulmonary, rectal, or dermal uptake.
- URP sequences that contain regions that are relatively rich in the positively charged amino acids arginine or lysine which favor cellular uptake or transport through membranes.
- URP sequences can be designed to contain one or several protease-sensitive sequences. Such URP sequences can be cleaved once the product of the invention has reached its target location. This cleavage may trigger an increase in potency of the pharmaceutically active domain (pro-drug activation) or it may enhance binding of the cleavage product to a receptor.
- URP sequences can be designed to carry excess negative charges by introducing aspartic acid or glutamic acid residues.
- URP that contain great than 5%, greater than 6%, 7%, 8%, 9%, 10%, 15%, 30% or more glutamic acid and less than 2% lysine or arginine.
- Such URPs carry an excess negative charge and as a result they have a tendency to adopt open conformations due to electrostatic repulsion between individual negative charges of the peptide.
- Such an excess negative charge leads to an effective increase in their hydrodynamic radius and as a result it can lead to reduced kidney clearance of such molecules.
- one can modulate the effective net charge and hydrodynamic radius of a URP sequence by controlling the frequency and distribution of negatively charged amino acids in the URP sequences.
- Most tissues and surfaces in a human or animal carry excess negative charges.
- URPs may have a repetitive amino acid sequence of the format (Motif) x in which a sequence motif forms a direct repeat (ie ABCABCABCABC) or an inverted repeat (ABCCBAABCCBA) and the number of these repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 50 or more.
- URPs or the repeats inside URPs often contain only 1, 2, 3, 4, 5 or 6 different types of amino acids.
- URPs typically consist of repeats of human amino acid sequences that are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36 or more amino acids long, but URPs may also consist of non-human amino acid sequences that are 20, 22, 24, 26, 28, 30, 32, 34 36, 38 40, 42, 44, 46, 48, 50 amino acids long.
- URPs can be derived from human sequences.
- the human genome contains many subsequences that are rich in one particular amino acid.
- amino acid sequences that are rich in a hydrophilic amino acid like serine, threonine, glutamate, aspartate, or glycine.
- subsequences that contain few hydrophobic amino acids.
- Such subsequences are predicted to be unstructured and highly soluable in aqeuous solution.
- Such human subsequences can be modified to further improve their utility.
- FIG. 17 shows an exemplary human sequence that is rich in serine and that can be isolated as the subject URP.
- the exemplified dentin sialophosphoprotein contains a 670-amino acid subsequence in which 64% of the residues are serine and most other positions are hydrophilic amino acids such as aspartate, asparagines, and glutamate.
- the sequence is extremely repetitive and as a result it has a low information content.
- Examples of sequences that are related to dentin sialophosphoprotein are (SSD) n , (SSDSSN) n , (SSE) n , where n is between about 4 and 200.
- sequences from human proteins is particularly desirable in design of URPs with reduced immunogenicity in a human subject.
- a key step for eliciting an immune response to a foreign protein is the presentation of peptide fragments of said protein by MHC class II receptors. These MHCII-bound fragments can then be detected by T cell receptors, which triggers the proliferation of T helper cells and initiates an immune response.
- T cell epitopes from pharmaceutical proteins has been recognized as a means to reduce the risk of eliciting an immune reaction (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108).
- MHCII receptors typically interact with an epitope having e.g., a 9-amino acid long region of the displayed peptides.
- an epitope having e.g., a 9-amino acid long region of the displayed peptides.
- Such oligomers have reduced risk of being immunogenic.
- the junction sequences between the monomer units can still contain T cell epitopes that can trigger an immune reaction, which is illustrated in FIG. 3 .
- the URP sequence in FIG. 2 designed as an oligomer based on multiple human sequences such that each 9mer subsequences of the oligomer can be found in a human protein. In these designs, every 9-mer subsequence is a human sequence.
- An example of a URP sequence based on three human sequences is shown in FIG. 5 .
- URP sequences based on a single human sequences such that all possible 9mer subsequences in the oligomeric URP sequences occur in the same human protein.
- An example is shown in FIG. 6 based on the POU domain that is rich in glycine and proline.
- the repeating monomer in the URP sequence is only a fragment of the human protein and its flanking sequences is identical to the repeating unit as illustrated in FIG. 6 .
- Non-oligomeric URP sequences can be designed based on human proteins as well.
- the primary conditions are that all 9mer sub-sequences can be found in human sequences.
- the amino acid composition of the sequences preferably contains few hydrophobic residues.
- URP sequences that are designed based on human sequences and that contain a large fraction of glycine residues.
- Host of interest can be any animals, including vertebrates and invertebrates.
- Preferred hosts are mammals such as primates (e.g. chimpanzees and humans), cetaceans (e.g. whales and dolphins), chiropterans (e.g. bats), perrisodactyls (e.g. horses and rhinoceroses), rodents (e.g. rats), and certain kinds of insectivores such as shrews, moles and hedgehogs.
- the URPs typically contain multiple copies of the repeat sequences or units, wherein the majority of segments comprising about 6 to about 15 contiguous amino acids are present in one or more native human proteins.
- majority of the segments refers to more than about 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, preferably 100%.
- each of the possible segments between about 6 to 15 amino acids, preferably between about 9 to 15 amino acids within the repeating units are present in one or more native human proteins.
- the URPs can comprise multiple repeating units or sequences, for example having 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeating units.
- URP sequences can be designed to be substantially free of epitopes recognized by human T cells. For instance, one can synthesize a series of semi-random sequences with amino acid compositions that favor denatured, unstructured conformations and evaluate these sequences for the presence of human T cell epitopes and whether they are human sequences. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences.
- T-cell epitopes This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and in particular 9-mer subsequences that are not human.
- An alternative is to evaluate multiple peptide sequences that can be assembled into repeating units as described in the previous section for the assembly of human sequences.
- Another alternative is to design URP sequences that result in low scores using epitope prediction algorithms like TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61).
- Another approach to avoiding T-cell epitopes is to avoid amino acids that can serve as anchor residues during peptide display on MHC, such as M, I, L, V, F.
- Hydrophobic amino acids and positively charged amino acids can frequently serve as such anchor residues and minimizing their frequency in a URP sequences reduces the chance of generating T-cell epitopes and thus eliciting an immune reaction.
- the selected URPs generally contain subsequences that are found in at least one human protein, and have a lower content of hydrophobic amino acids.
- URP sequences can be designed to optimize protein production. This can be achieved by avoiding or minimizing repetitiveness of the encoding DNA. URP sequences such as poly-glycine may have very desirable pharmaceutical properties but their manufacturing can be difficult due to the high GC-content of DNA sequences encoding for GRS and due to the presence of repeating DNA sequences that can lead to recombination.
- URP sequences can be designed to be highly repetitive at the amino acid level. As a result the URP sequences have very low information content and the risk of eliciting an immune reaction can be reduced.
- Non-limiting examples of URPs containing repeating amino acids are: poly-glycine, poly-glutamic acid, poly-aspartic acid, poly-serine, poly-threonine, (GX) n where G is glycine and X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 20, (GGX) n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 13, (GGGX) n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 10, (GGGGX) n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 8, (G 2 X) n where X is serine, aspartic acid, glutamic acid, threonine, or proline, n is at
- Products of the invention may contain URP sequences that are semi-random sequences. Examples are semi-random sequences containing at least 30, 40, 50, 60 or 70% glycine in which the glycines are well dispersed and in which the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 70, 60, 50, 40, 30, 20, or 10% when combined.
- a preferred semi-random URP sequence contains at least 40% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 10%.
- a more preferred random URP sequence contains at least 50% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 5%.
- URP sequences can be designed by combining the sequences of two or more shorter URP sequences or fragments of URP sequences. Such a combination allows one to better modulate the pharmaceutical properties of the product containing the URP sequences and it allows one to reduce the repetitiveness of the DNA sequences encoding the URP sequences, which can improve expression and reduce recombination of the URP encoding sequences.
- URP sequences can be designed and selected to possess several of the following desired properties: a) high genetic stability of the coding sequences in the production host, b) high level of expression, c) low (predicted/calculated) immunogenicity, d) high stability in presence of serum proteases and/or other tissue proteases, e) large hydrodynamic radius under physiological conditions.
- One exemplary approach to obtain URP sequences that meet multiple criteria is to construct a library of candidate sequences and to identify from the library the suitable subsequences. Libraries can comprise random and/or semi-random sequences. Of particular utility are codon libraries, which is a library of DNA molecules that contains multiple codons for the identical amino acid residue.
- Codon randomization can be applied to selected amino acid positions of a certain type or to most or all positions.
- True codon libraries encode only a single amino acid sequence, but they can easily be combined with amino acid libraries, which is a population of DNA molecules encoding a mixture of (related or unrelated) amino acids at the same residue position.
- Codon libraries allow the identification of genes that have relatively low repetitiveness at the DNA level but that encode highly repetitive amino acid sequences. This is useful because repetitive DNA sequences tend to recombine, leading to instability.
- codon libraries that encode limited amino acid diversity. Such libraries allow introduction of a limited number of amino acids in some positions of the sequence while other positions allow for codon variation but all codons encode the same amino acid.
- Such partially random oligonucleotides can be fused by overlap PCR or ligation-based approaches.
- Another method to generate libraries is to synthesize a starting sequence and subsequently subject said sequence to partial randomization.
- URP sequences with desirable properties can be identified from libraries using a variety of methods. Sequences that have a high degree of genetic stability can be enriched by cultivating the library in a production host. Sequences that are unstable will accumulate mutations, which can be identified by DNA sequencing. Variants of URP sequences that can be expressed at high level can be identified by screening or selection using multiple protocols known to someone skilled in the art. For instance one can cultivate multiple isolates from a library and compare expression levels.
- Expression levels can be measured by gel analysis, analytical chromatography, or various ELISA-based methods.
- the determination of expression levels of individual sequence variants can be facilitated by fusing the library of candidate URP sequences to sequence tags like myc-tag, His-tag, HA-tag.
- Another approach is to fuse the library to an enzyme or other reporter protein like green fluorescent protein.
- Of particular interest is the fusion of the library to a selectable marker like beta-lactamase or kanamycin-acyl transferase.
- One can use antibiotic selection to enrich for variants with high level of expression and good genetic stability.
- Variants with good protease resistance can be identified by screening for intact sequences after incubation with proteases.
- protease-resistant URP sequences is bacterial phage display or related display methods. Multiple systems have been described where sequences that undergo rapid proteolysis can be enriched by phage display. These methods can be easily adopted to enrich for protease resistant sequences. For example, one can clone a library of candidate URP sequences between an affinity tag and the pIII protein of M13 phage. The library can then be exposed to proteases or protease-containing biological samples like blood or lysosomal preparations. Phage that contain protease-resistant sequences can be captured after protease treatment by binding to the affinity tag.
- Phage display can be utilized to identify candidate URP sequences that do not bind to a particular immune serum in order to identify URP sequences with low immunogenicity.
- the resulting serum can then be used for phage panning to remove or identify sequences that are recognized by antibodies in the resulting immune serum.
- Other methods like bacterial display, yeast display, ribosomal display can be utilized to identify variants of URP sequences with desirable properties.
- URP sequences of interest Another approach is the identification of URP sequences of interest by mass spectrometry. For instance, one can incubate a library of candidate URP sequences with a protease or biological sample of interest and identify sequences that resist degradation by mass spectrometry. In a similar approach one can identify URP sequences that facilitate oral uptake. One can feed a mixture of candidate URP sequences to animals or humans and identify variants with the highest transfer or uptake efficiency across some tissue barrier (ie dermal, etc) by mass spectrometry. In a similar way, one can identify URP sequences that favor other uptake mechanisms like pulmonary, intranasal, rectal, transdermal delivery. One can also identify URP sequences that favor cellular uptake or URP sequences that resist cellular uptake.
- tissue barrier ie dermal, etc
- URP sequences can be designed by combining URP sequences or fragments of URP sequences that were designed by any of the methods described above.
- semi-random approaches to optimize sequences that were designed based on the rules described above.
- codon optimization is of particular importance for URP sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization can be performed using computer programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize ribosomal pausing (Coda Genomics Inc.).
- URP sequences When designing URP sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (ie the AGG and AGA arginine codons and one Leucine codon in E. coli ) DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels. Thus, when possible it is preferred to choose codons such that the GC-content of URP-encoding sequence is suitable for the production organism that will be used to manufacture the URP.
- URP encoding genes can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension.
- URP modules can be constructed such that the URP module-encoding gene has low repetitiveness while the encoded amino acid sequence has a high degree of repetitiveness. The approach is illustrated in FIG. 11 .
- a library of relatively short URP sequences This can be a pure codon library such that each library member has the same amino acid sequence but many different coding sequences are possible.
- To facilitate the identification of well-expressing library members one can construct the library as fusion to a reporter protein.
- reporter genes are green fluorescent protein, luciferace, alkaline phosphatase; beta-galactosidase.
- URP sequences contain particular amino acids at high fraction. Such sequences can be difficult to produce by recombinant techniques as their coding genes can contain repetitive sequences that are subject to recombination. Furthermore, genes that contain particular codons at very high frequencies can limit expression as the respective loaded tRNAs in the production host become limiting.
- An example is the recombinant production of GRS. Glycine residues are encoded by 4 triplets, GGG, GGC, GGA, and GGT. As a result, genes encoding GRS tend to have high GC-content and tend to be particularly repetitive. An additional challenge can result from codon bias of the production host. In the case of E.
- codon optimization of the gene encoding URP sequences can be very desirable.
- One can optimize codon usage by employing computer programs that consider codon bias of the production host (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53).
- codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members which are particularly suitable for the large-scale production of URP-containing products.
- MURPs Multivalent Unstructured Recombinant Proteins
- the subject URPs are particularly useful as modules for design of proteins of therapeutic value. Accordingly, the present invention provides proteins comprising one or more subject URPs. Such proteins are termed herein Multivalent Unstructured Recombinant Proteins (MURPs).
- MURPs Multivalent Unstructured Recombinant Proteins
- one or more URP sequences can be fused to the N-terminus or C-terminus of a protein or inserted in the middle of the protein, e.g., into loops of a protein or in between modules of the protein of interest, to give the resulting modified protein improved properties relative to the unmodified protein.
- the combined length of URP sequences that are attached to a protein can be 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- the subject MURPs exhibit one or more improved properties as detailed below.
- URP sequences to a pharmaceutically active protein can improve many properties of that protein.
- adding a long URP sequence can significantly increase the serum half-life of the protein.
- URPs typically contain amino acid sequences of at least about 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- the URPs can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs. Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 30 amino acids. Preferably, the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- the fused URPS can increase the hydrodynamic radius of a protein and thus reduces its clearance from the blood by the kidney. The increase in the hydrodynamic radius of the resulting fusion protein relative to the unmodified protein can be detected by ultracentrifugation, size exclusion chromatography, or light scattering.
- hydrophilic polymers have a tendency to accumulate selectively in tumor tissue which is caused by the enhanced permeability and retention (EPR) effect.
- EPR enhanced permeability and retention
- the underlying cause of the EPR effect is the leaky nature of tumor vasculature (McDonald, D. M., et al. (2002) Cancer Res, 62: 5381-5) and the lack of lymphatic drainage in tumor tissues. Therefore, the selectivity of pharmaceutically active proteins for tumor tissues can be enhanced by adding hydrophilic polymers.
- the therapeutic index of a given pharmaceutically active protein can be increased via incorporating the subject URPS.
- URP sequences can significantly improve the protease resistance of a protein.
- URP sequences themselves can be designed to be protease resistant and by attaching them to a protein one can shield that protein from the access of degrading enzymes.
- URP sequences can be added to pharmaceutically active proteins with the goal of reducing undesirable interactions of the protein with other receptors or surfaces. To achieve this, it can be beneficial to add the URP sequences to the pharmaceutically active protein in proximity to the site of the protein that makes such undesirable contacts.
- one can add URP sequences to pharmaceutically active proteins with the goal of reducing their interactions with any component of the immune system to prevent an immune response against the product of the invention.
- URP sequences can reduce interaction with pre-existing antibodies or B-cell receptors. Furthermore, the addition of URP sequences can reduce the uptake and processing of the product of the invention by antigen presenting cells. Adding one or more URP sequence to a protein is a preferred way of reducing its immunogenicity as it will suppress an immune response in many species allowing one to predict the expected immunogenicity of a product in patients based on animal data. Such species independent testing of immunogenicity is not possible for approaches that are based on the identification and removal of human T cell epitopes or sequences comparison with human sequences.
- URP sequences can be introduced into proteins in order to interrupt T cell epitopes. This is particularly useful for proteins that combine multiple separate functional modules.
- the formation of T cell epitopes requires that peptide fragments of a protein antigen bind to MHC. MHC molecules interact with a short segment of amino acids typically 9 contiguous residues of the presented peptides.
- the direct fusion of different binding modules in a protein molecule can lead to T cell epitopes that span two neighboring domains.
- URP modules By separating the functional modules by URP modules prevents the generation of such module-spanning T cell epitopes as illustrated in FIG. 7 .
- the insertion of URP sequences between functional modules can also interfere with proteolytic processing in antigen presenting cells, which will lead to an additional reduction of immunogenicity.
- Another approach to reduce the risk of immunogenicity is to disrupt T cell epitopes within functional modules of a product.
- one approach is to have some of the intercysteine loops (those that are not involved in target binding) be glycine-rich.
- microproteins whose structure is due to a small number of cysteines, one could in fact replace most or all of the residues that are not involved in target binding with glycine, serine, glutamate, threonine, thus reducing the potential for immunogenicity while not affecting the affinity for the target.
- this can be carried out by performing a ‘glycine-scan’ of all residues, in which each residue is replaced by a glycine, then selecting the clones which retain target binding using phage display or screening, and then combining all of the glycine substitutions that are permitted.
- functional modules have a much higher probability to contain T cell epitopes than URP modules.
- Functional modules of a protein can have limited solubility.
- binding modules tend to carry hydrophobic residues on their surface, which can limit their solubility and can lead to aggregation.
- URP modules By spacing or flanking such functional modules with URP modules one can improve the overall solubility of the resulting product. This is in particular true for URP modules that carry a significant percentage of hydrophilic or charged residues.
- By separating functional modules with soluble URP modules one can reduce intramolecular interactions between these functional modules
- URP sequences can be designed to carry an excess of negative or positive charges. As a result they confer an electrostatic field to any fusion partner which can be utilized to shift the pH profile of an enzyme or a binding interaction. Furthermore, the electrostatic field of a charged URP sequence can increase the homogeneity of pKa values of surface charges of a protein product, which leads to sharpened pH profiles of ligand interactions and to sharpened separations by isoelectric focusing or chromatofocusing.
- Each amino acid in solution by itself has a single, fixed pKa, which is the pH at which its functional groups are half protonated.
- pKa the pH at which its functional groups are half protonated.
- PEG is uncharged and does not affect the ionization spectrum of the protein it is attached to, leaving it with a broad ionization spectrum.
- a URP with a high content of Gly and Glu in principle exist in only two states: neutral (—COOH) when the pH is below the pKa of Glutamate and negatively charged (—COO ⁇ ) when the pH is above the pKa of Glutamate.
- URP modules can form a single, homogeneously ionizated type of molecule and can yield a single mass in mass spectrometry.
- MURPs can be expressed as a fusion with an URP having a single type of charge (Glu) distributed at constant spacing through the URP module.
- Glu type of charge
- the increase in the homogeneity of the charge of the protein population has favorable processing properties, such as in ion exchange, isoelectric focusing, mass spec, etc. compared to traditional PEGylation.
- URP sequences can be designed to be very hydrophilic and as a result they improve the solubility of (for example) human proteins, which often contain hydrophobic patches that they use to bind to other human proteins.
- the formulation of such human proteins, like antibodies, can be quite challenging and often limits their concentration and delivery options.
- URPs can reduce product precipitation and aggregation and it allows one to use simpler formulations containing fewer ingredients, that are typically needed to stabilize a product in solution.
- URP sequences-containing products allows to formulate these products at higher concentration and as a result one can reduce the injection volume for injectable products, which may enable home injection, which is limited to a very low injected volume. Addition of a URP sequence can also simplify the storage of the resulting formulated products.
- URP sequences can be added to pharmaceutically active proteins to facilitate their oral, pulmonary, rectal, or intranasal uptake. URP sequences can facilitate various modes of delivery because they allow higher product concentrations and improved product stability. Additional improvements can be achieved by designing URP sequences that facilitate membrane penetration.
- URP sequences can have significant benefits for the production of the resulting product.
- Many recombinant products, especially native human proteins have a tendency to form aggregates during production that can be difficult or impossible to dissolve and even when removed from the final product they may re-occur. These are usually due to hydrophobic patches by which these (native human) proteins contacted other (native human) proteins and mutating these residues is considered risky because of immunogenicity.
- URPs can increase the hydrophilicity of such proteins and enable their formulation without mutating the sequence of the human protein.
- URP sequences can facilitate the folding of a protein to reach its native state.
- Many pharmaceutically active proteins are produced by recombinant methods in a non-native aggregated state.
- URP-containing products are much easier to prepare as compared to polymer-modified proteins. Chemical polymer-modification requires extra modification and purification steps after the active protein has been purified. In contrast, URP sequences can be manufactured using recombinant DNA methods together with the pharmaceutically active protein.
- the products of the invention are also significantly easier to characterize compared to polymer-modified products.
- URP sequences can also facilitate the purification of a product.
- URP sequences can include subsequences that can be captured by affinity chromatography.
- An example are sequences rich in histidine, which can be captured on resins with immobilized metals like nickel.
- URP sequences can also be designed to have an excess of negatively or positively charged amino acids. As a result they can significantly impact the net charge of a product, which can facilitate product purification by ion-exchange chromatography or preparative electrophoresis.
- the subject MURPs can contain a variety of modules, including but not limited to binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.
- FIG. 1 depicts an exemplary MURP having multiple modules.
- MURPs can also have relatively simple architectures that are illustrated in FIG. 2 .
- MURPs can also contain fragmentation sites. These can be protease-sensitive sequences or chemically sensitive sequences that can be preferentially cleaved when the MURPs reach their target site.
- Binding module refers to a peptide or protein sequence that can bind specifically to one or several targets, which may be one or more therapeutic targets or accessory targets, such as for cell-, tissue- or organ targeting.
- BMs can be linear or cyclic peptides, cysteine-constrained peptides, microproteins, scaffold proteins (e.g., fibronectin, ankyrins, crystalline, streptavidin, antibody fragments, domain antibodies), peptidic hormones, growth factors, cytokines, or any type of protein domain, human or non-human, natural or non-natural, and they may be based on a natural scaffold or not based on a natural scaffold, or based on combinations or they may be fragments of any of the above.
- these BMs can be engineered by adding, removing or replacing one or multiple amino acids in order to enhance their binding properties, their stability, or other properties.
- Binding modules can be obtained from natural proteins, by design or by genetic package display, including phage display, cellular display, ribosomal display or other display methods. Binding modules may bind to the same copy of the same target, which results in avidity, or they may bind to different copies of the same target (which can result in avidity if these copies are somehow connected or linked, such as by a cell membrane), or they may bind to two unrelated targets (which yields avidity if these targets are somehow linked, such as by a membrane). Binding modules can be identified by screening or otherwise analyzing random libraries of peptides or proteins.
- the Tepitope score of a protein is the log of the Kd (dissociation constant, affinity, off-rate) of the binding of that protein to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 15 logs, from about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, -4, -5 (10e 10 Kd) to about ⁇ 5.
- Preferred MURPs yield a score less than about ⁇ 3.5 [KKW: On absolute scale?]
- binding modules comprising disulfide bonds formed by pairing two cysteine residues.
- the binding modules comprise polypeptides having high cysteine content or high disulfide density (HDD).
- HDD high disulfide density
- Binding modules of the HDD family typically have 5-50% (5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50%) cysteine residues and each domain typically contains at least two disulfides and optionally a co-factor such as calcium or another ion.
- HDD scaffold allows these modules to be small but still adopt a relatively rigid structure. Rigidity is important to obtain high binding affinities, resistance to proteases and heat, including the proteases involved in antigen processing, and thus contributes to the low or non-immunogenicity of these modules.
- the disulfide framework folds the modules without the need for a large number of hydrophobic side chain interactions in the interior of most modules.
- the small size is also advantageous for fast tissue penetration and for alternative delivery such as oral, nasal, intestinal, pulmonary, blood-brain-barrier, etc. In addition, the small size also helps to reduce immunogenicity.
- a higher disulfide density is obtainable, either by increasing the number of disulfides or by using domains with the same number of disulfides but fewer amino acids. It is also desirable to decrease the number of non-cysteine fixed residues, so that a higher percentage of amino acids is available for target binding.
- the cysteine-containing binding modules can adopt a wide range of disulfide bonding patterns (DBPs).
- DBPs disulfide bonding patterns
- two-disulfide modules can have three different disulfide bonding patterns (DBPs)
- three-disulfide modules can have 15 different DBPs
- four-disulfide modules have up to 105 different DBPs.
- the total number of disulfide bonding patterns can be calculated according to the formula:
- ⁇ i 1 n ⁇ ⁇ 2 ⁇ i - 1 ,
- n the predicted number of disulfide bonds formed by the cysteine residues
- ⁇ represents the product of (2i-1), where i is a positive integer ranging from 1 up to n.
- the modules used in MURPs are natural or non-naturally occurring cysteine (C)-containing scaffold exhibiting a binding specificity towards a target molecule, wherein the non-naturally occurring cysteine (C)-containing scaffold comprise intra-scaffold cysteines according to a pattern selected from the group of permutations represented by the formula
- ⁇ i 1 n ⁇ ⁇ 2 ⁇ i - 1 ,
- the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having two disulfide bonds formed by pairing cysteines contained in the polypeptide according to a pattern selected from the group consisting of C 1-2, 3-4 , C 1-3, 2-4 , and C 1-4, 2-3 , wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond.
- the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having three disulfide bonds formed by pairing intra-scaffold cysteines according to a pattern selected from the group consisting of C 1-2, 3-4, 5-6 , C 1-2, 3-5, 4-6 , C 1-2, 3-6, 4-5 , C 1-3, 2-4, 5-6 , C 1-3, 2-5, 4-6 , C 1-3, 2-6, 4-5 , C 1-4, 2-3, 5-6 , C 1-4, 2-6, 3-5 , C 1-5, 2-3, 4-6 , C 1-5, 2-4, 3-6 , C 1-5, 2-6, 3-4 , C 1-6, 2-3, 4-5 , and C 1-6, 2-5, 3-4 , wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond.
- the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having at least four disulfide bonds formed by pairing cysteines contained in the polypeptide according to a pattern selected from the group of permutations defined by the formula above.
- the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having at least five, six, or more disulfide bonds formed by pairing intra-protein cysteines according to a pattern selected from the group of permutations represented by the formula above. Any of the cysteine-containing proteins or scaffolds disclosed in the co-pending application Ser. Nos. 11/528,927 and 11/528,950, which are incorporated herein by reference in their entiety] are candidate binding modules.
- Binding modules can also be selected from libraries of cysteine-constrained cyclic peptides with 4, 5, 6, 7, 8, 9, 10, 11 and 12 randomized or partially randomized amino acids between the disulfide-bonded cystines (e.g., in a build-up manner), and in some cases additional randomized amino acids on the outside of the cystine pair can be constructed using a variety of methods.
- Library members with specificity for a target of interest can be identified using various methods including phage display, ribosomal display, yeast display and other methods known in the art.
- Such cyclic peptides can be utilized as binding modules in MURPs.
- One particular buildup approach is illustrated in FIG. 25 . It is based on the addition of a single cysteine plus multiple randomized residues on the N-terminal side of the previously selected cyclic peptide, as well as on the C-terminal side.
- Such buildup libraries can contain between 1 and 12 random positions on the N-terminal as well as on the C-terminal side of a cyclic peptide.
- the distance between the cysteine residues in the newly added random flanks and the cysteine residues in the cyclic peptide can be varied between 1 and 12 residues.
- Such buildup approaches can be performed with clone-specific primers so that it leaves no fixed sequence between the library areas as shown in FIG.
- FIG. 26 can be applied to a collection of cyclic peptides with specificity for a target of interest. Both buildup approaches were shown to work for anti-VEGF affinity maturation by build-up. This approach can be repeated to generate binding modules with six or more cysteine residues.
- FIG. 27 Another buildup of a one-disulfide into a 2-disulfide sequence is illustrated in FIG. 27 . It involves the dimerization of a previously selected pool of 1-disulfide peptides with itself so that the preselected peptide pool ends up in the N-terminal as well as in the C-terminal position. This approach favors the build up of 2-disulfide sequences that recognize two separate epitopes on a target.
- Another buildup approach involves the addition of a (partially) randomized sequence of 6-15 residues containing two cysteines that are spaced 4, 5, 6, 7, 8, 9, or 10 amino acids apart, with optionally additional randomized positions outside the linked cysteines.
- This 2-cysteine random sequence is added on the N-terminal side of the previously selected peptide, or on the C-terminal side.
- This approach favors a 1-2 3-4 DBP, although other DBPs may be formed.
- This approach can be repeated to generate binding modules with six or more cysteine residues.
- Binding modules can be constructed based on natural protein scaffolds. Such scaffolds can be identified by data base searching. Libraries that are based on natural scaffolds can be subjected to phage display panning followed by screening to identify sequences that specifically bind to a target of interest.
- a wide selection of natural scaffolds is available for constructing the binding modules.
- the choice of a particular scaffold will depend on the intended target.
- Non-limiting examples of natural scaffolds include snake-toxin-like proteins such as snake venom toxins and extracellular domain of human cell surface receptors.
- Non-limiting examples of snake venom toxins are Erabutoxin B, gamma-Cardiotoxin, Faciculin, Muscarininc toxin, Erabutoxin A, Neurotoxin I, Cardiotoxin V4II (Toxin III), Cardiotoxin V, alpha-Cobratoxin, long Neurotoxin 1, FS2 toxin, Bungarotoxin, Bucandin, Cardiotoxin CTXI, Cardiotoxin CTX IIB, Cardiotoxin II, Cardiotoxin III, Cardiotoxin IV, Cobrotoxin 2, alpha-toxins, Neurotoxin II (cobrotoxin B), Toxin B (long neurotoxin), Candotoxin, Bucain.
- snake venom toxins are Erabutoxin B, gamma-Cardiotoxin, Faciculin, Muscarininc toxin, Erabutoxin A, Neurotoxin I, Cardiotoxin V4
- Non-limiting examples of extracellular domain of (human) cell surface receptors include CD59, Type II activin receptor, BMP receptor Ia ectodomain, TGF-beta type II receptor extracellular domain.
- Other natural scaffolds include but are not limited to A-domains, EGF, Ca-EGF, TNF-R, Notch, DSL, Trefoil, PD, TSP1, TSP2, TSP3, Anato, Integrin Beta, Thyroglobulin, Defensin 1, Defensin 2, Cyclotide, SHKT, Disintegrins, Myotoxins, Gamma-Thioneins, Conotoxin, Mu-Conotoxin, Omega-Atracotoxins, Delta-Atracotoxins, as well as additional families disclosed in co-pending application Ser. Nos. 11/528,927 and 11/528,950, which are incorporated herein in their entirety.
- a large variety of methods has been described that allow one to identify binding molecules in a large library of variants.
- One method is chemical synthesis.
- Library members can be synthesized on beads such that each bead carries a different peptide sequence.
- Beads that carry ligands with a desirable specificity can be identified using labeled binding partners.
- Another approach is the generation of sub-libraries of peptides which allows one to identify specific binding sequences in an iterative procedure (Pinilla, C., et al. (1992) BioTechniques, 13: 901-905). More commonly used are display methods where a library of variants is expressed on the surface of a phage, protein, or cell.
- Phage typically carry 3-5 copies of this protein and thus phage in such a library will in most cases carry between 3-5 copies of a library member. This approach is referred to as multivalent display.
- An alternative is phagemid display where the library is encoded on a phagemid. Phage particles can be formed by infection of cells carrying a phagemid with a helper phage. (Lowman, H. B., et al. (1991) Biochemistry, 30: 10832-10838). This process typically leads to monovalent display. In some cases, monovalent display is preferred to obtain high affinity binders. In other cases multivalent display is preferred (O'Connell, D., et al. (2002) J Mol Biol, 321: 49-56).
- phage display A variety of methods have been described to enrich sequences with desirable characteristics by phage display.
- a phage library is contacted with the immobilized target, phage that lack a binding ligand are washed away, and phage carrying a target specific ligand can be eluted by a variety of conditions. Elution can be performed by low pH, high pH, urea or other conditions that tend to break protein-protein contacts.
- Bound phage can also be eluted by adding E. coli cells such that eluting phage can directly infect the added E. coli host.
- protease which can degrade the phage-bound ligand or the immobilized target.
- Proteases can also be utilized as tools to enrich protease resistant phage-bound ligands. For instance, one can incubate a library of phage-bound ligands with one or more (human or mouse) proteases prior to panning on the target of interest. This process degrades and removes protease-labile ligands from the library (Kristensen, P., et al. (1998) Fold Des, 3: 321-8). Phage display libraries of ligands can also be enriched for binding to complex biological samples.
- Random mixtures of nucleotides can be utilized to synthesize oligonucleotides that contain one or multiple random positions. This process allows one to control the number of random positions as well as the degree of randomization. In addition, one can obtain random or semi-random DNA sequences by partial digestion of DNA from biological samples. Random oligonucleotides can be used to construct libraries of plasmids or phage that are randomized in pre-defined locations. This can be done by PCR fusion as described in (de Kruif, J., et al. (1995) J Mol Biol, 248: 97-105).
- Kunkel mutagenesis uses templates containing randomly incorporated uracil bases which can be obtained from E. coli strains like CJ236.
- the uracil-containing template strand is preferentially degraded upon transformation into E. coli while the in vitro synthesized mutagenized strand is retained.
- most transformed cells carry the mutagenized version of the phagemid or phage.
- a valuable approach to increase diversity in a library is to combine multiple sub-libraries. These sub-libraries can be generated by any of the methods described above and they can be based on the same or on different scaffolds.
- Another variation of the Scholle method is to design the mutagenic oligonucleotide such that an amber stop codon in the template is converted into an ochre stop codon, and an ochre into an amber in the next cycle of mutagenesis.
- the template phage and the mutagenized library members must be cultured in different suppressor strains of E. coli , alternating an ochre suppressor with amber suppressor strains. This allows one to perform successive rounds of mutagenesis of a phage by alternating between these two types of stop codons and two suppressor strains.
- the megaprimer is a long ssDNA that was generated from the library inserts of the selected pool of phage from the previous round of panning.
- the goal is to capture the full diversity of library inserts from the previous pool, which was mutagenized in one or more areas, and transfer it to a new library in such a way that an additional area can be mutagenized.
- the megaprimer process can be repeated for multiple cycles using the same template which contains a stop-codon in the gene of interest.
- the megaprimer is a ssDNA (optionally generated by PCR) which contains 1) 5′ and 3′ overlap areas of at least 15 bases for complementarity to the ssDNA template, and 2) one or more previously selected library areas (1, 2, 3, 4 or more) which were copied (optionally by PCR) from the pool of previously selected clones, and 3) a newly mutagenized library area that is to be selected in the next round of panning.
- the megaprimer is optionally prepared by 1) synthesizing one or more oligonucleotides encoding the newly synthesized library area and 2) by fusing this, optionally using overlap PCR, to a DNA fragment (optionally obtained by PCR) which contains any other library areas which were previously optimized.
- Run-off or single stranded PCR of the combined (overlap) PCR product is used to generate the single stranded megaprimer that contains all of the previously optimized areas as well as the new library for an additional area that is to be optimized in the next panning experiment.
- This approach is expected to allow affinity maturation of proteins using multiple rapid cycles of library creation generating 10e11 to 10e12 diversity per cycle, each followed by panning.
- a variety of methods can be applied to introduce sequence diversity into (previously selected or na ⁇ ve) libraries of microproteins or to mutate individual microprotein clones with the goal of enhancing their binding or other properties like manufacturing, stability or immunogenicity.
- all the methods that can be used to generate libraries can also be used to introduce diversity into enriched (previously selected) libraries of microproteins.
- one can synthesize variants with desirable binding or other properties and design partially randomized oligonucleotides based on these sequences. This process allows one to control the positions and degree of randomization.
- One can deduce the utility of individual mutations in a protein from sequence data of multiple variants using a variety of computer algorithms (Jonsson, J., et al.
- PCR conditions have been described that lead to mutations at relatively high mutation frequencies (Leung, D., et al. (1989) Technique, 1: 11-15).
- a polymerase with reduced fidelity can be employed (Vanhercke, T., et al. (2005) Anal Biochem, 339: 9-14).
- a method of particular interest is based on mutator strains (Irving, R. A., et al. (1996) Immunotechnology, 2: 127-43; Coia, G., et al. (1997) Gene, 201: 203-9). These are strains that carry defects in one or more DNA repair genes. Plasmids or phage or other DNA in these strains accumulate mutations during normal replication.
- the libraries can be further treated to reduce artifacts.
- Known artifacts of phage panning include 1) no-specific binding based on hydrophobicity, and 2) multivalent binding to the target, either due to a) the pentavalency of the pIII phage protein, or b) due to the formation of disulfides between different microproteins, resulting in multimers, or c) due to high density coating of the target on a solid support and 3) context-dependent target binding, in which the context of the target or the context of the microproteins becomes critical to the binding or inhibition activity.
- Different treatment steps can be taken to minimize the magnitude of these problems. For example, such treatments are applied to the whole library, but some useful treatments that remove bad clones can only be applied to pools of soluble proteins or only to individual soluble proteins.
- cysteine-containing scaffolds are likely to contain free thiols, which can complicate directed evolution by cross-linking to other proteins.
- One approach is to remove the worst clones from the library by passing it over a free-thiol column, thus removing all clones that have one or more free sulfhydryls.
- Clones with free SH groups can also be reacted with biotin-SH reagents, enabling efficient removal of clones with reactive SH groups using Streptavidin columns.
- Another approach is to not remove the free thiols, but to inactivate them by capping them with sulfhydryl-reactive chemicals such as iodoacetic acid.
- sulfhydryl-reactive chemicals such as iodoacetic acid.
- bulky or hydrophilic sulfhydryl reagents that reduce the non-specific target binding or modified variants.
- context dependence are all of the constant sequences, including pIII protein, linkers, peptide tags, biotin-streptavidin, Fc and other fusion proteins that contribute to the interaction.
- the typical approach for avoiding context-dependence involves switching the context as frequently as practical in order to avoid buildup. This may involve alternating between different display systems (ie M13 versus T7, or M13 versus Yeast), alternating the tags and linkers that are used, alternating the (solid) support used for immobilization (ie immobilization chemistry) and alternating the target proteins itself (different vendors, different fusion versions).
- Library treatments can also be used to select for proteins with preferred qualities.
- One option is the treatment of libraries with proteases in order to remove unstable variants from the library.
- the proteases used are typically those that would be encountered in the application.
- For pulmonary delivery one would use lung proteases, for example obtained by a pulmonary lavage.
- mixtures of single purified proteases An extensive list of proteases is shown in [Appendix E]. The phage themselves are exceptionally resistant to most proteases and other harsh treatments.
- reducing agent ie DTT or betamercaptoethanol
- concentrations from 2.5 mM, to 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or even 100 mM, depending on the desired stability.
- Filter lift is another methodology that can be with bacterial colonies grown at high density on large agar plates (10e2-10e5). Small amounts of some proteins are secreted into the media and end up bound to the filter membrane (nitrocellulose or nylon). The filters are then blocked in non-fat milk, 1% Casein hydrolysate or a 1% BSA solution and incubated with the target protein that has been labeled with a fluorescent dye or an indicator enzyme (directly or indirectly via antibodies or via biotin-streptavidin). The location of the colony is determined by overlaying the filter on the back of the plate and all of the positive colonies are selected and used for additional characterization.
- the advantage of filter lifts is that it can be made to be affinity-selective by reading the signal after washing for different periods of time.
- affinity characterization typically requires a 3-point assay with a well-based assay and may provide better clone-to-clone comparability than well-based assays. Gridding of colonies into an array is useful since it minimizes differences due to colony size or location.
- the subject MURPs can contain N-terminal modules (NM), which are particularly useful e.g., in facilitating production of the MURPs.
- NM N-terminal modules
- the NM can be a single methionine residue when the products is expressed in the E. coli cytoplasm.
- a typical product format is an URP fused to a therapeutic protein, which is expressed in the bacterial cytoplasm so that the N-terminus is formyl-methionine.
- the formyl-methionine can either be permanent or temporary, if it is removed by biological or chemical processing.
- the NM can also be a peptide sequence that has been engineered for proteolytic processing, which can be used to remove tags or to remove fusion proteins.
- the N-terminal module can be engineered to facilitate the purification of the MURP by including an affinity tag such as the Flag-, Myc-, HA- or His-tag.
- the N-terminal module can also include an affinity tag that can be used for the detection of the MURP.
- An NM can be engineered or selected for high-level expression of the MURP. It can also be engineered or selected to enhance the protease resistance of the resulting MURP.
- MURPs can be produced with an N-terminal module that facilitates expression and/or purification. This N-terminal module can be cleaved off during the production process with a protease, such that the final product does not contain an N-terminal module.
- the N-terminal module can also contain a processing site that can be cleaved by a specific protease like factor Xa, thrombin, or enterokinase, Tomato Etch Virus (TEV) protease. Processing sites can also be designed to be cleavable by chemical hydrolysis.
- An example is the amino acid sequence asp-pro that can be cleaved under acidic conditions.
- An N-terminal module can also be designed to facilitate the purification of a MURP.
- N-terminal modules can be designed to contain multiple his residues which allow product capture by immobilized metal chromatography.
- N-terminal modules can contain peptide sequences that can be specifically captured or detected by antibodies. Examples are FLAG, HA, c-myc.
- MURPs can contain a C-terminal module, which are particularly useful e.g., in facilitating production of the MURPs.
- C-terminal module can comprise a cleavage site to effect proteolytic processing to remove sequences that are fused and hence increasing protein expression or facilitating purification.
- the C-terminal module can also contain a processing site that can be cleaved by a specific protease like factor Xa, thrombin, TEV protease or enterokinase. Processing sites can also be designed to be cleavable by chemical hydrolysis.
- An example is the amino acid sequence asp-pro that can be cleaved under acidic conditions.
- the C-terminal module can be an affinity tag aimed at facilitating the purification of the MURP.
- C-terminal modules can be designed to contain multiple his residues which allow product capture by immobilized metal chromatography.
- C-terminal modules can contain peptide sequences that can be specifically captured or detected by antibodies.
- Non-limiting examples of the tags include FLAG-, HA-, c-myc, or His-tag.
- C-terminal module can also be engineered or selected to enhance the protease resistance of the resulting MURP.
- the N-terminus of the protein can be linked to its own C-terminus.
- linking these two modules can be carried out by creating an amino acid-like natural linkage (peptide bond) or by using an exogenous linking entity.
- peptide bond amino acid-like natural linkage
- exogenous linking entity e.g. cyclotides, a family of small proteins in which this occurs naturally. Adopting a structural format like cyclotides is expected to provide additional stability against exo-proteases. Such intramolecular linkage typically works better at lower protein concentrations.
- MURPs can comprise one or multiple effector modules (EMs), or none at all. Effector modules typically do not provide the targeting, but they provide an activity required for therapeutic effect, like cell-killing.
- EMs can be pharmaceutically active small molecules (ie toxic drugs), peptides or proteins. Non-limiting examples are cytokines, antibodies enzymes, growth factors, hormones, receptors, receptor agonists or antagonists, whether whole or a fragment or domain thereof.
- Effector modules can also comprise peptide sequences that carry chemically linked small molecule drugs, whether synthetic or natural.
- these effector molecules can be linked to the effector module via chemical linkers, which may or may not be cleaved under selected conditions leading to a release of the toxic activity.
- EMs can also include radioisotopes and their chelates, as well as various labels for PET and MRI.
- Effector modules can also be toxic to a cell or a tissue.
- MURPs that contain toxic effector modules and binding modules with specificity for a diseased tissue or disease cell type. Such MURPs can specifically accumulate in a diseased tissue or in diseased cells and the can exert their toxic action preferentially in the diseased cells or tissues.
- exemplary effector modules are exemplary effector modules.
- Enzymes can be enzymes. Of particular interest are enzymes that degrade metabolites that are critical for cellular growth like carbohydrates or amino acids or lipids or co-factors. Other examples for effector modules with enzymatic activity are RNase, DNase, and phosphatase, asparaginase, histidinase, arginase, betalactamase. Effector modules with enzymatic activity can be toxic when delivered to a tissue or cell. Of particular interest are MURPs that combine effector modules that are toxic and binding modules that bind specifically to a diseased tissue. Enzymes that convert an inactive prodrug into an active drug at the tumor site are also potential effector modules.
- the subject MURP can contain an effector that is a drug.
- sequences can be designed for the organ-selective delivery of drug molecules.
- FIG. 8 An example is illustrated in FIG. 8 .
- An URP sequence can be fused to a protein that preferentially binds to diseased tissue.
- the same URP sequence can contain one or more amino acid residues that can be modified for the attachment of drug molecules.
- Such a conjugate can bind to diseased tissue with high specificity and the attached drug molecules can result in local action while minimizing systemic drug exposure.
- the MURP can be designed to facilitate the release of drug molecules at the target size by introducing protease-sensitive sites that can be cleaved by native proteases at the site of desired action.
- URP sequences for the design of drug delivery constructs.
- Many drug molecules that can be conjugated to targeting domains have significant hydrophobicity and the resulting conjugates tend to aggregate.
- hydrophilic URP sequences to such constructs one can improve the solubility of the resulting delivery constructs and as a consequence reduce the aggregation tendency.
- the use of URP sequences allows one to optimize the distance between the drug conjugation sites to facilitate complete conjugation.
- chemotherapeutic agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmus
- paclitaxel (TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERETM, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-1); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives
- chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin, doxorubicin, daunomycin, duocarmycin, vincristin, and vinblastin.
- anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (
- drugs that can be used as the effector modules include those that are useful for treating inflammatory conditions, cardiac diseases, infectious diseases, respiratory diseases, autoimmune diseases, neronal and muscular disorders, metabolic disorders, and cancers.
- Additional drugs that can be used as the effectors in MURPs include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are generated by biotransformation of the products of the selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory agents, analgesic-antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of the inducible cyclooxygenase, selective inhibitors of the inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, ⁇ -adren
- drugs that can be used as effector include agents for the treatment of peptic ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetics, agents used in irritable bowel syndrome, agents used for diarrhea, agents used for constipation, agents used for inflammatory bowel disease, agents used for biliary disease, agents used for pancreatic disease.
- Radionuclides can be designed for the tissue-targeted delivery of radionuclides as well as for imagin with radionuclides.
- URPs are ideal for imaging because the halflife can be optimized by changing the length of the URP. For most imaging applications a moderately long URP is likely to be preferred, providing a halflife of 5 minutes to a few hours, not days or weeks MURPs can be designed such that they only contain a single or a small defined number of amino groups that can be modified with chelating agents (such as DOTA) for radio isotopes such as technetium, indium, yttrium, (EXPAND). Alternative methods of conjugation are through reserved cysteine side chains.
- chelating agents such as DOTA
- EXPAND chelating agents
- Alternative methods of conjugation are through reserved cysteine side chains.
- Such radionuclide-carrying MURPs can be employed for the treatment of tumors or other diseased tissues, as well as for imaging.
- cytokines cytokines, growth factors, enzymes, -receptors, microproteins, hormones, erythopoetin, adenosine deiminase, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, rasburicase, axokine, RNAse, DNAse, phosphatase, pseudomonas exotoxin, ricin, gelonin, desmoteplase, laronidase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor IIV, factor VIII, factor X, factor IX, dornase, glucocere
- Protease-activated MURPs To enhance the therapeutic index of an effector module, one can insert protease-labile sequences into URP sequences that are sensitive to proteases that are preferentially found in serum or in the target tissue to be treated by the MURP. This approach is illustrated in FIG. 9 . Some designs allows one to construct proteins that are selectively activated when reaching a target tissue. Of particular interest are MURPs that are activated at a disease site. To facilitate such target-specific activation one can attach URP sequences in close proximity to the active site or receptor binding site of the effector module such that the resulting fusion protein has limited biological activity. Of particular interest is the activation of an effector module at a tumor site.
- PSA prostate specific antigen
- Prodrugs consisting of a PSA-labile peptide conjugated to the cancer drug doxorubicin have shown selective activation in prostate tissue [DeFeo-Jones, D., et al. (2000) Nat Med, 6: 1248].
- proteins with cytostatic or cytotoxic activity like TNFalpha, and many cytokines and interleukins.
- Another application is the selective activation of proteins at the site of inflammation or at site of virus or bacterial infection.
- MURPs containing URP sequences can be produced using molecular biology approaches that are well know in the art.
- a variety of cloning vectors are available for various expression systems like mammalian cells, yeast, and microbes.
- expression hosts are E. coli, S. cerevisiae, P. pastoris , and chinese hamster ovary cells.
- hosts that have been optimized to widen their codon usage.
- a host that has been modified to enhance expression of GRS That can be done by providing DNA that encodes glycine-specific tRNAs.
- the DNA encoding the enhanced protein can be operationally linked to a promoter sequences.
- the DNA encoding the enhanced protein as well as the operationally linked promoter can be part of a plasmid vector, viral vector or it can be inserted into the chromosome of the host.
- For production on can culture the host under conditions that facilitate the production of the enhanced protein.
- Conditions that improve the production of GRS are conditions that improve the production of GRS.
- the subject MURPs can adopt a variety of formats.
- the MURPs can contain URPs that are fused to pharmaceutically active proteins to produce slow-release products. Such products can be injected or implanted locally for instance into or under the skin of a patient. Due to its large hydrodynamic radius the URP sequences-containing product is slowly released from the injection or implantation site which leads to a reduction of the frequency of injection or implantation.
- the URP sequences can be designed to contain regions that bind to cell surfaces or tissue in order to prolong the local retention of the drug at the injection site.
- URP-containing products that can be formulated as soluble compounds but form aggregates or precipitates upon injection.
- This aggregation or precipitation can be triggered by a change in pH between the formulated product and the pH at the injection site.
- URP-containing products that precipitate or form aggregates as a result of a change in redox conditions.
- URP-containing product that is stabilized in solution by addition of non-active solutes, but that precipitates or aggregates upon injection as a result of diffusion of the solubilizing solutes.
- Another approach is to design URP-containing products that contain one or multiple Lysine or Cysteine residues in their URP sequence and that can be cross-linked prior to injection.
- the MURP is monomeric (here meaning not-crosslinked) when manufactured and formulated and when injected, but after subcutaneous injection the protein starts to crosslink with itself or with native human proteins, forming a polymer under the skin from which active drug molecules are freed only very gradually.
- Such release can be by disulfide bond reduction or disulfide shuffling as illustrated in FIG. 18 , or it can be mediated by proteolysis as shown in FIG. 19 , releasing active fragments into the circulation. It is important that these active fragments are large enough to have a long halflife, because the longer their secretion halflife, the lower the dose of the released protein can be, allowing the use of a lower dose of product to be injected or a longer time between injections.
- a protein drug would be manufactured with a cyclic peptide in it (one or more). This cyclic peptide may or may not be involved in binding to the target. This protein is manufactured with the cyclic peptide formed, ie in oxidized form, to simplify purification. However, the product is then reduced and formulated to keep the protein in reduced form.
- the cyclic peptide reduces at a low concentration of reducing agent, such as 0.25, 0.5, 1.0, 2.0, 4.0 or 8.0 mM Dithiothreitol or Betamercaptoethanol or cysteine or equivalent reducing agent, so that the cyclic peptide can be reduced without reducing other disulfide containing protein modules in the product.
- reducing agent such as 0.25, 0.5, 1.0, 2.0, 4.0 or 8.0 mM Dithiothreitol or Betamercaptoethanol or cysteine or equivalent reducing agent.
- FDA approved reducing agents is preferred, such as cysteine or glutathione.
- the low molecular weight reducing agent diffuses away rapidly or is neutralized by human proteins, exposing the drug to an oxidizing environment while it is still at a high molar concentration, which causes crosslinking of cysteines located on different protein chains, which leads to polymerization of the drug at the injection site.
- disulfide reduction and oxidation will cause disulfide reshuffling, which will lead to cyclic peptide reformation and monomerization and resolubilization of the drug.
- the release of the drug from the polymer can also occur via proteolysis which could be targeted and controlled or increased by building in cleavage sites for serum proteases.
- the crosslinking to the proteins could also be performed with a chemical protein-protein crosslinking agent, such as the ones listed in [table x]. Ideally, this is an already FDA-approved agent, such as those used for vaccine conjugation or conjugation of chemicals to proteins.
- disulfides instead of using disulfides, one can also stabilize proteins against proteolytic degradation using a wide variety of crosslinking agents. Most of the agents below are sold by Pierce Chemicals under that same name and instructions for their use are available online (www.piercenet.com). The agents that result in the same chain-to-chain distance as obtained with disulfides are the most likely to be useful for this application. The short-linker agents such as DFDNB are the most promising. The interchain distance can be readily determined from the structures of the chemicals as shown in www.piercenet.com.
- crosslinking agents are Imidoesters, active halogens, maleimide, pyridyl disulfide, NHS-ester.
- Homobifunctional crosslinking agents have two identical reactive groups and are often used in a onestep chemical crosslinking procedure.
- BS3 a non-cleavable water-soluble DSS analog
- BSOCOES base-reversible
- DMA Dimethyl adipimidate-2HCl
- DMP Dimethyl pimelimidate-2HCl
- DMS Dimethyl suberimidate-2HCl
- DSG 5-carbon analog of DSS
- DSP Limant's reagent
- DSS non-cleavable
- DST cleavable by oxidizing agents
- DTBP Dimethyl 3,3′-dithiobispropionimidate-2HCl
- DTSSP EGS, Sulfo-EGS, THPP, TSAT
- DFDNB 1,5-Difluoro-2,4-dinitrobenzene
- Sulfhydryl-reactive homobifunctional crosslinking agents are homobifunctional protein crosslinkers that react with sulfhydryls are often based on maleimides, which react with —SH groups at pH 6.5-7.5, forming stable thioether linkages.
- BM[PEO]3 is an 8-atom polyether spacer that reduces potential for conjugate precipitation in sulfhydryl-to-sulfhydryl cross-linking applications.
- BM[PEO]4 is similar but with an 11-atom spacer.
- BMB is a non-cleavable crosslinker with a four-carbon spacer. BMDB makes a linkage that can be cleaved with periodate.
- BMH is a widely used homobifunctional sulfhydryl-reactive crosslinker.
- BMOE has an especially short linker.
- DPDPB and DTME are cleavable crosslinkers.
- HVBS does not have the hydrolysis potential of meleimides.
- TMEA is another option.
- Hetero-bifunctional crosslinking agents have two different reactive groups. Examples are NHS-esters and amines/hydrazines via EDC activation, AEDP, ASBA (photoreactive, iodinatable), EDC (water-soluble carbodiimide).
- Amine-Sulflhydryl reactive bifunctional crosslinkers are AMAS, APDP, BMPS, EMCA, EMCS, GMBS, KMUA, LC-SMCC, LC-SPDP, MBS, SBAP, SIA (extra short), SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-LC-SMPT, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB.
- Amino-group reactive heterobifunctional crosslinking agents are ANB-NOS, MSA, NHS-ASA, SADP, SAED, SAND, SANPAH, SASD, SFAD, Sulfo-HSAB, Sulfo-NHS-LC-ASA, Sulfo-SADP, Sulfo-SANPAH, TFCS.
- a different slow release format has the drug labeled with a His6 tag, which is mixed and co-injected with Nickel-Nitrilotriacetic acid-conjugated beads (Ni-NTA beads), a GMO version of the ones that are available from Qiagen.
- the drug would slowly teach off the beads, providing depot and slow release as illustrated in FIG. 20 .
- the beads are optional and can be replaced by a crosslinked, polymeric Nickel-nitrilotriacetic acid that leads to assembly of an even larger polymer.
- URP sequences can contain sequences that are known to form multimers like alpha2D [Hill, R., et al. (1998) J Am Chem Soc, 120: 1138-1145] that was utilized to dimerize an antibody fragment [Kubetzko, S., et al. (2005) Mol Pharmacol, 68: 1439-54].
- Examples of a useful homo dimerization peptide is the sequence SKVILFE.
- An example of useful heterodimerization sequences are the peptide ARARAR that can form dimers with the sequence DADADA and related sequences. Multimerization can improve the biological function of a molecule by increasing its avidity and it can influence pharmacokinetic properties and tissue distribution of the resulting MURPs.
- Multimerization modules are amino acid sequences that facilitate dimer or multimer formation of MURPs. Multimerization modules may bind to themselves to form dimers or multimers. Alternatively, multimerization modules can bind to other modules of the MURP. These can be leucine zippers or small peptides like Hydra head activator derivatives (SKVILF-like) which forms antiparallel homopolymers, or peptides like RARARA and DADADA, which form high affinity antiparallel heteropolymers. Using one, two or more copies of these peptides one can force the formation of protein dimers, linear multimers or branched multimers.
- SKVILF-like Hydra head activator derivatives
- the affinity of the association can be tailored by changing the type, length and composition of the peptides. Some applications require peptides that form homodimers as illustrated in FIG. 21 . Other applications require heterodimers. In some cases, once associated, the peptides can be locked into place by forming disulfide bonds between the two protein chains, typically on either side of the peptides. Multimerization modules are useful for linking two MURP molecules together (head to tail, head to head, or tail to tail) as illustrated in FIG. 21 . The multimerization modules can be located on either the N- or C-terminus in order to form dimers. If the multimerization modules are present at both termini, long, linear multimers will be formed.
- branched polymeric networks can be formed.
- the concepts of multimerization and chemical conjugation can be combined leading to useful for halflife extension and depot formation, leading to slow release of active drug from the depot or injection site as illustrated in FIG. 23 .
- the subject MURPs can incorporate a genetic or universal URP.
- One approach is to express a URP containing a long URP module, which provides halflife and contains multiple (typically 4-10) lysines (or other sites) that allows site-specific conjugation of peptides (ie linear, cyclic, 2SS, 3 SS, etc) that bind to a specific target.
- the advantage of this approach is that the URP module is generic and can be conjugated with any target-specific peptide.
- the linkage of the target-specific peptide to the URP is a directed linkage, so that residues on the URP can only react with a residue on the target-specific peptide and exhaustive coupling can only produce a single species, which is a URP that is linked to a peptide at every lysine, for example.
- This complex behaves like a high-avidity multimer in it's binding properties but is simple to manufacture. This approach is illustrated in FIG. 24 .
- the subject MURPs can also incorporate URPs to effect delivery across tissue barriers.
- URPs can be engineered to enhance delivery across the dermal, oral, buccal, intestinal, nasal, blood-brain, pulmonary, thecal, peritoneal, rectal, vaginal or many other tissue barriers.
- URP sequences can improve protease resistance of pharmaceutically active proteins and thus facilitate their uptake. It has been shown that protein uptake in the digestive system can be improved by adding molecular carriers. The main role of these carriers is an improvement of membrane permeability [Stoll, B. R., et al. (2000) J Control Release, 64: 217-28]. Thus one can include sequences into URP sequences that improve membrane permeability. Many sequences that improve membrane permeability are know and examples are sequences rich in arginine [Takenobu, T., et al. (2002) Mol Cancer Ther, 1: 1043-9].
- URP sequences that improve cellular or oral uptake of proteins by combining two functions, a reduction in proteolytic degradation of the protein of interest as well as an increase in membrane permeability of the fusion product.
- a sequence to the URP sequence that is sensitive to a protease that is preferentially located at in the target tissue for the drug of interest but is stable to proteases in the digestive tract.
- Examples of such URP sequences are sequences that contain long regions of GRS as well as sequences that are rich in basic amino acids in particular arginine and facilitate membrane transfer.
- URP can be utilized in a similar way to improve protein uptake via intranasal, intrapulmonary, or other routes of delivery.
- DR4/DR5 agonist DR4 and DR5 are death receptors that are expressed on many tumor cells. These receptors can be triggered by trimerization which leads to cell death and tumor regression. Binding domains with specificity for DR4 or DR5 can be obtained by phage panning or other display methods. These DR4 or DR5-specific binding domains can be multimerized using URP modules as linkers as illustrated in FIG. 12 . Of particular interest are MURPs that contain three or more binding modules with specificity for DR4 or DR5 or both. As illustrated in FIG. 12 , MURPs can contain additional binding modules with specificity for tumor antigens that are overexpressed in tumor tissues. This allows one to construct MURPs that specifically accumulate in tumor tissue and trigger cell death. MURPs can contain modules that bind either DR4 or DR5. Of particular interest are MURPs that contain binding modules that bind both DR4 and DR5.
- Interleukin 2 is a cytokine that can enhance the immune response to tumor tissue.
- systemic IL2 therapy is characterized by significant side effects.
- MURPs can be constructed that combine binding domains with specificity for tumor antigens and IL2 as effector module as illustrated in FIG. 13 .
- Such MURP can selectively accumulate in tumor tissue and thus elicit a tumor-selective immune response while minimizing the systemic side effects of cytokine therapy.
- Such MURPs can target a variety of tumor antigens like EpCAM, Her2, CEA, EGFR, Thomsen Friedenreich antigen.
- MURPs that bind to tumor antigens that show slow internalization. Similar MURPs can be designed using other cytokines or tumor necrosis factor-alfa as effector modules.
- Tumor-selective asparaginase is used to treat patients with acute leukemia. Both asparaginase from E. coli and asparaginase from Erwinia are used for treatment. Both enzymes can lead to immunogenicity and hypersensitive reactions. Oncaspar is PEGylated version of asparaginase that has reduced immunogenicity. However, the protein is difficult to manufacture and administered as a mixture of isomers. Adding URP sequences to termini and/or to internal loops allows the direct recombinant manufacture of an asparaginase variant that is homogeneous and has low immunogenicity. Various URP sequences and attachment sites can be compared to determine the optimum position for URP sequence attachment.
- the subject MURPs can be designed to target any cellular proteins. A non-limiting list is provided below.
- VEGF vascular endothelial growth factor
- human target proteins that are commercially available in purified form. Examples are: 4EBP1, 14-3-3 zeta, 53BP1, 2B4/SLAMF4, CCL21/6Ckine, 4-1BB/TNFRSF9, 8D6A, 4-1BB Ligand/TNFSF9, 8-oxo-dG, 4-Amino-1,8-naphthalimide, A2B5, Aminopeptidase LRAP/ERAP2, A33, Aminopeptidase N/ANPEP, Aag, Aminopeptidase P2/XPNPEP2, ABCG2, Aminopeptidase P1/XPNPEP1, ACE, Aminopeptidase PILS/ARTS1, ACE-2, Amnionless, Actin, Amphiregulin, beta-Actin, AMPK alpha 1/2, Activin A, AMPK alpha 1, Activin AB, AMPK alpha 2, Activin B, AMPK beta 1, Activin C,
- Non-limiting examples include 5-hydroxytryptamine 3 receptor B subunit, 5-hydroxytryptamine 3 receptor precursor, 5-hydroxytryptamine receptor 3 subunit C, AAD14 protein, Acetylcholine receptor protein, alpha subunit precursor, Acetylcholine receptor protein, beta subunit precursor, Acetylcholine receptor protein, delta subunit precursor, Acetylcholine receptor protein, epsilon subunit precursor, Acetylcholine receptor protein, gamma subunit precursor, Acid sensing ion channel 3 splice variant b, Acid sensing ion channel 3 splice variant c, Acid sensing ion channel 4, ADP-ribose pyrophosphatase, mitochondrial precursor, Alpha1A-voltage-dependent calcium channel, Amiloride-sensitive cation channel 1, neuronal, Amiloride-sensitive cation channel 2, neuronal Amiloride-sensitive cation channel 4, isoform 2, Amiloride-sensitive sodium channel,
- Exemplary GPCRs include but are not limited to Class A Rhodopsin like receptors such as Musc. acetylcholine Vertebrate type 1, Musc. acetylcholine Vertebrate type 2, Musc. acetylcholine Vertebrate type 3, Musc.
- Adrenoceptors (Alpha Adrenoceptors type 1, Alpha Adrenoceptors type 2, Beta Adrenoceptors type 1, Beta Adrenoceptors type 2, Beta Adrenoceptors type 3, Dopamine Vertebrate type 1, Dopamine Vertebrate type 2, Dopamine Vertebrate type 3, Dopamine Vertebrate type 4, Histamine type 1, Histamine type 2, Histamine type 3, Histamine type 4, Serotonin type 1, Serotonin type 2, Serotonin type 3, Serotonin type 4, Serotonin type 5, Serotonin type 6, Serotonin type 7, Serotonin type 8, other Serotonin types, Trace amine, Angiotensin type 1, Angiotensin type 2, Bombesin, Bradykinin, C5a anaphylatoxin, Finet-leu-phe, APJ like, Interleukin-8 type A, Interleukin-8 type B, Interleukin
- the subject MURPs can be designed to target any cellular proteins including but not limited to cell surface protein, secreted protein, cytosolic protein, and nuclear protein.
- a target of particular interest is an ion channel.
- Ion channels constitute a superfamily of proteins, including the family of potassium channels (K-channels), the family of sodium channels (Na-channels), the family of calcium channels (Ca-channels), the family of Chlorine channels (Cl-channels) and the family of acetylcholine channels.
- K-channels potassium channels
- Na-channels sodium channels
- Ca-channels calcium channels
- Cl-channels Chlorine channels
- acetylcholine channels acetylcholine channels.
- K-channel family contains subfamilies of voltage-gated K-channels called Kv1.x and Kv3.x.
- Kv1.x contains the channels Kv1.1, Kv1.2 and Kv1.3, which correspond to the products of single genes and are thus called ‘species’.
- the classification applies to the Na-, Ca-, Cl- and other families of channels as well.
- Ion channels can also be classified according to the mechanisms by which the channels are operated. Specifically, the main types of ion channel proteins are characterized by the method employed to open or close the channel protein to either permit or prevent specific ions from permeating the channel protein and crossing a lipid bilayer cellular membrane.
- One important type of channel protein is the voltage-gated channel protein, which is opened or closed (gated) in response to changes in electrical potential across the cell membrane.
- the voltage-gated sodium channel 1.6 (Nav1.6) is of particular interest as a therapeutic target.
- Another type of ion channel protein is the mechanically gated channel, for which a mechanical stress on the protein opens or closes the channel.
- Still another type is called a ligand-gated channel, which opens or closes depending on whether a particular ligand is bound to the protein.
- the ligand can be either an extracellular moiety, such as a neurotransmitter, or an intracellular moiety, such as an ion or nucleotide.
- Ion channels generally permit passive flow of ions down an electrochemical gradient, whereas ion pumps use ATP to transport against a gradient. Coupled transporters, both antiporters and symporters, allow movement of one ion species against its gradient, powered by the downhill movement of another ion species.
- potassium channel proteins One of the most common types of channel proteins, found in the membrane of almost all animal cells, permits the specific permeation of potassium ions across a cell membrane.
- potassium ions permeate rapidly across cell membranes through K + channel proteins (up to 10 ⁇ 8 ions per second).
- potassium channel proteins have the ability to distinguish among potassium ions, and other small alkali metal ions, such as Li + or Na + with great fidelity.
- potassium ions are at least ten thousand times more permanent than sodium ions.
- Potassium channel proteins typically comprise four (usually identical) subunits, so their cell surface targets are present as tetramers, allowing tetravalent binding of MURPs.
- One type of subunit contains six long hydrophobic segments (which can be membrane-spanning), while the other types contains two hydrophobic segments.
- HVA channels comprises at least three groups of channels, known as L-, N- and P/Q-type channels. These channels have been distinguished one from another electrophysiologically as well as bio-chemically on the basis of their pharmacology and ligand binding properties. For instance, dihydropyridines, diphenyl-alkylamines and piperidines bind to the ⁇ 1 subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents.
- N-type calcium channels are sensitive to omega conopeptides, but are relatively insensitive to dihydropyridine compounds, such as nimodipine and nifedipine.
- P/Q-type channels are insensitive to dihydropyridines, but are sensitive to the funnel web spider toxin Aga IIIA.
- R-type calcium channels like L-, N-, P- and Q-type channels, are activated by large membrane depolarizations, and are thus classified as high voltage-activated (HVA) channels.
- HVA high voltage-activated
- R-type channels are generally insensitive to dihydropyridines and omega conopeptides, but, like P/Q, L and N channels, are sensitive to the funnel web spider toxin AgaIVA.
- Neuronal voltage-sensitive calcium channels typically consists of a central ⁇ 1 . subunit, an ⁇ 2 / ⁇ subunit, a ⁇ subunit and a 95 kD subunit.
- Kir an inwardly rectified potassium channel
- Kv a voltage-gated potassium channel
- Nav a voltage-gated sodium channel
- Cav a voltage-gated calcium channel
- CNG cyclic nucleotide-gated channel
- HCN hyperpolarization-activated channel
- TRP a transient receptor potential channel
- ClC a chloride channel
- CFTR a cystic fibrosis transmembrane conductance regulator
- RYR a ryanodine receptor
- Other channel types are 2-pore channels, glutamate-receptors (AMPA, NMDA, KA), M2, Connexins and Cys-loop receptors.
- TRPC1 and TRPC5 are to have six membrane-spanning segments, arranged as follows:
- S1-6 are membrane-spanning sequences
- E1-3 are extracellular surface loops
- X1-2 are intracellular surface loops.
- the E3 loop is generally the longest of the three extracellular loops and is hydrophilic so it is a good target for drugs and MURPs to bind.
- the pore-forming part of most channels is a multimeric (e.g. tetrameric or rarely pentameric) complex of membrane-spanning alpha-helices.
- the ion channels are valuable targets for drug design because they are involved in a broad range of physiological processes.
- ion channel proteins many of which have been implicated in genetic diseases.
- abbrebrant expression or function of ion channels has been shown to cause a wide arrange of diseases including cardiac, neuronal, muscular, respiratory metabolic diseases.
- This section focuses on ion channels, but the same concepts and approaches are equally applicable to all membrane proteins, including 7TMs, 1TMs, G-proteins and G-Protein Coupled receptors (GPCRs), etc.
- GPCRs G-Protein Coupled receptors
- Ion channels typically form large macromolecular complexes that include tightly bound accessory protein subunits and combinatorial use of such subunits contributes to the diversity of ion channels.
- accessory proteins can also be the binding targets of the subject MURPs, microproteins and toxins.
- MURPs can be designed to bind any of the channels known in the art and to those specifically exemplified herein.
- MURPs exhibiting a desired ion channel binding capability can be selected by any recombinant and biochemical (e.g. expression and display) techniques known in the art.
- MURPs can be displayed by a genetic package including but not limited to phages and spores, and be subjected to panning against intact cell membranes, or preferably intact cells such as whole mammalian cells. To remove the phage that bind to the other, non-target cell surface molecules, the standard approach was to perform subtraction panning against similar cell lines that had a low or non-detectable level of the target receptor.
- Popkov et al. J. Immunol. Methods 291:137-151 (2004) showed that related cell types are not ideal for subtraction because they generally have a reduced but still significant level of the target on their surface, which reduces the number of desired phage clones. This problem occurs even when panning on cells that have been transfected with the gene encoding the target, followed by negative selection/subtraction on the same cell-line which was not transfected, especially when the native target gene was not knocked out. Instead, Popkov et al.
- negative selection or subtraction panning works much better if performed with an excess of the same cells that are used for normal panning (positive selection), except that the target has now been blocked with a high-affinity, target-specific inhibitor, such as a small molecule, peptide or an antibody to the target, which makes the active site unavailable.
- a high-affinity, target-specific inhibitor such as a small molecule, peptide or an antibody to the target, which makes the active site unavailable.
- This process is called “negative selection with epitope-masked cells”, which is particularly useful in selecting the subject MURPs with a desired ion-channel binding capability.
- the present invention provides microproteins, and particularly microproteins exhibiting binding capability towards at least one family of ion channels.
- the present invention also provides a genetic package displaying such microproteins.
- Non-limiting ion-channel examples to which the subject microproteins bind are sodium, potassium, calcium, acetylcholine, and chlorine channels.
- Native targets are generally natural molecules or fragments, derivatives thereof that the microprotein is known to bind, typically including those known binding targets that have been reported in the literature.
- the subject invention also provides a genetic package displaying an ion-channel-binding microprotein which has been modified.
- the modified microprotein may (a) binds to a different family of channel as compared to the corresponding unmodified microprotein; (b) binds to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein; (c) binds to a different species of the same subfamily of channel as compared to the corresponding unmodified microprotein; (d) the microprotein binds to a different site on the same channel as compared to the corresponding unmodified microprotein; and/or (e) binds to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein.
- FIGS. 22 and 46 show how microprotein domains or toxins that each bind at different sites of the same ion channel can be combined into a single protein.
- the two binding sites that these two microproteins bind to can be on two channels from different families, two channels from the same family but a different subfamily, two channels from the same subfamily but a different species (gene product), or two different binding sites on the same channel (species) or they can (simultaneously or not) bind the same binding site on the same channel (species) since the channels are multimeric.
- the binding modules and domains that bind to sites on the channels can be microprotein domains (natural or non-natural, 2- to 8-disulfide containing), one-disulfide peptides, or linear peptides. These modules can be selected independently and combined, or one can be selected from a library to bind in the presence of one fixed, active binding module. In the latter case, the display library would display multiple modules of which one would contain a library of variants. A typical goal is to select a dimer from this library that has a higher affinity than the active monomer that was the starting point.
- the present invention provides a protein comprising a plurality of ion-channel binding domains, wherein individual domains are microprotein domains that have been modified such that (a) the microprotein domains bind to a different family of channel as compared to the corresponding unmodified microprotein domains; (b) the microprotein domains bind to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein domains; (c) the microprotein domains bind to a different species of the same subfamily as compared to the corresponding unmodified microprotein domains; (d) the microprotein domains bind to a different site on the same channel as compared to the corresponding unmodified microprotein domains; (e) the microprotein domains bind to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein domains; and/or (f) the microprotein domains bind to the same site of the same channel and yield the same biological effect as compared to the corresponding unmodified micro
- the microprotein domains may comprise natural or non-natural sequences.
- the individual domains can be linked together via a heterologous linker.
- the individual microprotein domains can bind to the same or different channel family, same or different channel subfamily, same or different species of the same subfamily, same or different site on the same channel.
- the subject microproteins can be a toxin.
- the toxin retains in part or in whole its toxicity spectrum.
- venomous animals such as snakes, encounter a range of prey and intruder species and the venom toxins differ in activity for the different receptors of the different species.
- the venom consists of a large number of related and unrelated toxins, with each toxin having a “spectrum of activity”, which can be defined as all of the receptors from all of the species on which that toxin has measurable activity. All of the targets in the ‘spectrum of activity’ are considered “native targets” and this includes any human targets that the toxin is active against.
- the native target(s) of a microprotein or toxin include all of the targets that the toxin is reported to inhibit in the literature. The higher the affinity or activity on a target, the more likely that target is the natural, native target, but it is not uncommon for toxins to act on multiple targets within the same species.
- Native target(s) can be human or non-human receptors that the toxin is active against.
- the toxin may be desirable to test both the N-terminus and C-terminus for fusion and to test a variety of fusion sites (i.e., 0, 1, 2, 3, 4, 5, 6 amino acids before the first cysteine or after the last cysteine of the toxin domain, if the toxin domain is a cysteine-containing domain) using a synthetic DNA library approach, preferably encoding a library of glycine-rich linkers, which form the smallest amino acid chain, are uncharged and are most likely to be compatible with binding of the toxin to the target.
- a synthetic DNA library approach preferably encoding a library of glycine-rich linkers, which form the smallest amino acid chain, are uncharged and are most likely to be compatible with binding of the toxin to the target.
- the library should contain a lysine or a arginine to mimic the positively charged amino group (or fusions to the N-terminus of the toxin) and a glutamate or an aspartate to mimic the negatively charged carboxyl group (for fusions to the C-terminus of the toxin).
- the inhibitor(s) that are used to block the target during negative selection can be small molecules, peptides or proteins, and natural or non-natural.
- the choice of the mixture of inhibitors is a valuable tool to control the specificity of the ion channel inhibitors that are being designed. Because there are over three hundreds ion channels in total, with partially overlapping specificities and sequence similarities, and multiple modulatory sites per channel, each having a different effect, the specificity requirement can be complex.
- the two toxins When modifying the activity of a toxin, or when combining two different toxins into a single protein, the two toxins can bind the same channel at the same site and have the same physiologic effect, or the two toxins can bind the same channel at the same site and have a different physiologic effect, or the two toxins can bind to the same channel at a different site, or the two toxins can bind to different channels that belong to the same subfamily (i.e. Kv1.3 and Kv1.2; meaning product of a different gene or ‘species’), or the two toxins can bind to different channels that belong to the same family (i.e. both are K-channels), or the two toxins can bind to channels that belong to different families (i.e. K-channels versus Na-channels).
- the same subfamily i.e. Kv1.3 and Kv1.2; meaning product of a different gene or ‘species’
- the two toxins can bind to different channels that belong to the same family (i.e.
- Ion channels typically have many transmembrane segments (24 for sodium channels) and thus offer a number of different, non-competing and non-overlapping binding sites for modulators to alter the activity of the channel in different ways.
- One approach is to create binders for one site on the same ion channel from existing binders for a different site, even if these sites are unrelated.
- the existing toxin can be used as a targeting agent for a library of 1-, 2-, 3-, or 4-disulfide proteins that is separated from the targeting toxin by a flexible linker of 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40 or 50 amino acids.
- Another approach is to create new modulators for channels from existing modulators for other channels that are related in sequence or in structure.
- the conotoxin family contains sequence-related and structure-related modulators for Ca-, K, Na-channels and nicotinic acetylcholine receptors. It appears feasible to convert a K-channel modulator into a Na-channel modulator using a library of conotoxin-derivatives, or vice versa.
- Kappa-conotoxins inhibit K-channels
- Mu-conotoxins and Delta-conotoxins inhibit Na-channels
- Omega-conotoxins inhibit Ca-channels
- Alpha-conotoxins inhibit acetylcholine receptors.
- the individual modules can be panned and selected separately, in isolation of each other, or they can be designed in each other's presence, such that the new domain is added to a display system as a library that also contain a fixed, active copy that serves as a targeting element for the library and only clones that are significantly better than the fixed, active monomer are selected and characterized.
- FIGS. 46 and 47 show some of the monomeric derivatives that can be made from native (natural) toxins, and some of the multimers that can be made to bind at multiple different binding sites of the target.
- the linkers are shown as glycine-rich rPEG, but the linkers could be any sequence and could also be optimized using molecular libraries followed by panning.
- Such libraries can be based on existing toxins with known activity for that site, or they can be or naive 1-, 2-, 3-, 4-disulfide libraries based on unrelated microprotein scaffolds. These additional contact elements can be added on one or both sides of the active domains, and can be directly adjacent to the existing modulatory domain or they can be separated from it by flexible linkers.
- the initial multimer or the final, improved multimer can be a homomultimer or a heteromultimer, based on sequence similarity of the domains or based on target specificity of the domains of the multimer.
- the monomers that comprise the multimer may bind to the same target sites but have the same or different sequences.
- the binding capability of the subject MURPs, microproteins, or toxins to a given ion channel can be measured in terms of Hill Coefficient.
- Hill Coefficient indicates the stoichiometry of the binding interaction.
- a Hill coefficient of 2 indicates that 2 inhibitors bind to each channel.
- the biological activity or effect of an ion channel and the ability of the subject MURPs, microproteins or toxins to regulate an ion channel activity can be assessed using a variety of in vitro and in vivo assays. For instance, methods are available in the art for measuring voltage, measuring current, measuring membrane potential, measuring ion flux, e.g., potassium or rubidium, measuring ion concentration, measuring gating, measuring second messengers and transcription levels, and using e.g., voltage-sensitive dyes, radioactive tracers, and patch-clamp electrophysiology. In particular such assays can be used to test for microproteins and toxins that can inhibit or activate an ion channel of interest.
- potential channel inhibitors or activators can be tested in comparison to a suitable control to examine the extent of modulation.
- Control samples can also be samples untreated with the candidate activators or inhibitors. Inhibition is present when a given ion channel activity value relative to the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or even less.
- IC50 is a commonly used unit (the concentration of inhibitor that reduces the ion channel's activity by 50%) for determining the inhibitory effect. Similar for IC90.
- Activation of channels is achieved when the select a given ion channel activity value relative to the control is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more.
- Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing the channel of interest. For instance, one method is to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al., Pflugers. Archiv. 391:85 (1981).
- the effects of the candidate MURPs, microproteins, or toxins upon the function of a channel of interest can be measured by changes in the electrical currents or ionic flux or by the consequences of changes in currents and flux.
- the downstream effect of the candidate proteins on ion flux can be varied. Accordingly, any suitable physiological change can be used to assess the influence of a candidate protein on the test channels.
- the effects of candidate protein can be measured by a toxin binding assay.
- transmitter release e.g., dopamine
- hormone release e.g., insulin
- transcriptional changes to both known and uncharacterized genetic markers e.g., northern blots
- cell volume changes e.g., in red blood cells
- immunoresponses e.g., T cell activation
- changes in cell metabolism such as cell growth or pH changes
- changes in intracellular second messengers such as Ca2 + .
- ion selectivity is the ability of some channels to discriminate between ion species, allowing some to pass through the pore while excluding others.
- Gating is the transition between open and closed states. They can be assessed by any of the methods known in the art or disclosed herein
- Gating Frequency is influenced by voltage (in voltage gated channels, which are opened or closed by changes in membrane voltage) and ligand-binding.
- the transition rate between open and closed states is typically ⁇ 10 microseconds but can be increased or decreased by other molecules.
- the flux rate (current) through the pore when it is open is on the order of 10e7 ions per second for ion channels and much less for coupled exchangers.
- some voltage-gated channels enter an inactivated, non-conducting state in which they are refractory to depolarization.
- the human genome data base was searched for sequences that are rich in glycine. Three sequences were identified as suitable donor sequences as shown in Table X.
- a glycine rich sequence that contains multiple repeats of the peptide A with sequence GGGSGSGGGGS.
- Peptide A can be oligomerized to form structures with the formula (GGGSGSGGGGS) n where n is between 2 and 40.
- FIG. 5 shows that all possible 9mer subsequences in oligomers of peptide A are contained in at least one of the proteins listed in table 3. Thus oligomers of peptide A do not contain human T cell epitopes. Inspection of FIG. 5 reveals that GRS based on oligomers of peptide A can begin and end at any of the positions of peptide A.
- Glycine rich sequences were designed based on sequence GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG, which represents amino acids 146-182 of the human class 4 POU domain with accession number NP — 006228.
- FIG. 6 illustrates that oligomers of peptide B with sequence GGGGGPGGGGP can be utilized as GRS. All 9mer subsequences that are contained in peptides with the sequence (GGGGGPGGGGP) n are also contained in the sequence of the POU domain. Thus, such oligomeric sequences do not contain T cell epitopes.
- Glycine rich sequences can be designed based on the subsequence GAGGEGGGGEGGGPGG that is part of the ribosomal protein S6 kinase (accession number BAD92170). For instance, oligomers of peptide C with the sequence GGGGE will form sequences where most 9mer subsequences will be contained in the sequence of ribosomal protein S6 kinase. Thus, oligomeric GRS of the general structure (GGGGE) n bear a very low risk of containing T cell epitopes.
- a data base of human proteins was searched for subsequences that are rich in glycine residues. These subsequences contained at least 50% glycine. Only the following non-glycine residues were allowed to occur in the GRS: ADEHKPRST. 70 subsequences were identified that had a minimum length of 20 amino acids. These subsequences are listed in appendix A. They can be utilized to construct GRS with low immunogenic potential in humans.
- the following example describes the construction of a codon optimized gene encoding a URP sequence with 288 amino acids and the sequence (GSGGEG) 48 .
- a stuffer vector pCW0051 as illustrated in FIG. 40 .
- the sequence of the expression cassette in pCW0051 is shown in FIG. 42 .
- the stuffer vector was based on a pET vector and includes a T7 promoter.
- the vector encodes a Flag sequence followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites.
- the BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion as illustrated in FIG. 42 .
- the stuffer sequence was followed by a His 6 tag and the gene of green fluorescent protein (GFP).
- the stuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCW0051 formed non-fluorescent colonies.
- the stuffer vector pCW0051 was digested with BsaI and KpnI.
- a codon library encoding URP sequences of 36 amino acid length was constructed as shown in FIG. 41 .
- the URP sequence was designated rPEG_J36 and had the amino acid sequence (GSGGEG) 6 .
- the insert was obtained by annealing synthetic oligonucleotide pairs encoding the amino acid sequence GSGGEGGSGGEG as well as a pair of oligonucleotides that encode an adaptor to the KpnI site.
- the following oligonucleotides were used: pr_LCW0057 for: AGGTAGTGGWGGWGARGGWGGWTCYGGWGGAGAAGG, pr_LCW0057rev:
- the annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of rPEG_J12 repeats.
- the product corresponding to the length of rPEG_J36 was isolated from the mixture by agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0051.
- the isolates were pooled and dimerized using a process outlined in FIG. 40 .
- a plasmid mixture was digested with BsaI/NcoI and a fragment comprising the rPEG_J36 sequence and a part of GFP was isolated.
- the same plasmid mixture was also digested with BbsI/NcoI and the vector fragment comprising rPEG_J36, most of the plasmid vector, and the remainder of the GFP gene was isolated. Both fragments were mixed, ligated, and transformed into BL21 and isolates were screened for fluorescence. This process of dimerization was repeated two more rounds as outlined in FIG. 14 .
- rPEG_H288 A library of genes encoding a 288 amino acid URP termed rPEG_H288 was constructed using the same procedure that was used to construct rPEG_J288.
- rPEG_H288 has the amino acid sequence (GSGGEGGSGGSG) 24 .
- the flow chart of the construction process in shown in FIG. 14 .
- a fusion protein containing the an N-terminal Flag tag and the URP sequence rPEG_J288 fused to the N-terminus of green fluorescent protein was incubated in 50% mouse serum at 37 C for 3 days. Samples were withdrawn at various time points and analyzed by SDS PAGE followed by detection using Western analysis. An antibody against the N-terminal flag tag was used for Western detection. Results are shown in FIG. 28 , which indicate that a URP sequence of 288 amino acids can be completely stable in serum for at least three days.
- a gene encoding human interferon alpha was designed using codon optimization for E. coli expression.
- the synthetic gene was fused with a gene encoding rPEG_J288.
- a His6 tag was placed at the N-terminus to facilitate detection and purification, of the fusion protein.
- the amino acid sequence of the fusion protein is given in FIG. 44 .
- a gene encoding human G-CSF was designed using codon optimization for E. coli expression.
- the synthetic gene was fused with a gene encoding rPEG_J288.
- a His6 tag was placed at the N-terminus to facilitate detection and purification of the fusion protein.
- the amino acid sequence of the fusion protein is given in FIG. 44 .
- a gene encoding human growth hormone was designed using codon optimization for E. coli expression.
- the synthetic gene was fused with a gene encoding rPEG_J288.
- a His6 tag was placed at the N-terminus to facilitate detection and purification of the fusion protein.
- the amino acid sequence of the fusion protein is given in FIG. 44 .
- the fusion proteins between rPEG_J288 and two human proteins, interferon-alpha and human growth hormone were cloned into a T7 expression vector and transformed into E. coli BL21.
- the cells were grown at 37 C to an optical density of 0.5 OD. Subsequently, the cells were cultured at 18 C for 30 min. Then 0.5 mM IPTG was added and the cultures were incubated in a shaking incubator at 18 C overnight. Cells were harvested by centrifugation and soluble protein was released using BugBuster (Novagen). Both, insoluble and soluble protein fractions were separated by SDS-PAGE and the fusion proteins were detected by Western using and antibody against the N-terminal His6 tag for detection.
- FIG. 45 shows the Western analysis of the two fusion proteins as well as rPEG_J288-GFP as control. All fusion proteins were expressed and the majority of the protein was in the soluble fraction. This is evidence of the high solubility of rPEG_J288 because most attempts at expression of the interferon-alpha and human growth hormone in the cytosol of E. coli , that have been reported in the literature, resulted in the formation of insoluble inclusion bodies.
- FIG. 45 shows that the majority of fusion proteins are expressed as full length proteins, i.e. no fragments that would suggest incomplete synthesis or partial protein degradation were detected.
- the fusion sequence was dimerized using restriction enzymes and ligation steps to construct a molecule that contains 4 copies of the VEGF binding module separated by rPEG_A36 fused to GFP.
- the VEGF binding affinity of fusion proteins containing between zero and four VEGF-binding units were compared in FIG. 30 .
- a fusion protein containing only rPEG_A36 fused to GFP shows no affinity for VEGF. Adding increasing numbers of VEGF binding modules increases affinity of the resulting fusion proteins.
- Random peptide libraries were generated according to Scholle, et al. [Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51].
- the na ⁇ ve peptide libraries displayed cysteine-constrained peptides with cysteines spaced by 4 to 10 random residues.
- the library design is illustrated in the table:
- the libraries were panned agains a series of therapeutically relevant targets using the following protocol:
- Wells on immunosorbent ELISA plates were coated with 5 ⁇ g/ml of the target antigen in PBS overnight at 4° C. Coated plates were washed with PBS, and non-specific sites were blocked with Blocking Buffer (PBS containing either 0.5% BSA or 0.5% Ovalbumin) for 2 h at room temperature.
- the plates were then washed with PBST (PBS containing 0.05% Tween 20), and phage particles at 1-5 ⁇ 10 12 /ml in Binding Buffer (Blocking Buffer containing 0.05% Tween 20) were added to the wells and incubated with shaking for 2 h at room temperature.
- Blocking Buffer PBS containing either 0.5% BSA or 0.5% Ovalbumin
- ELISA plates were prepared by coating antigen and control proteins (BSA, Ovalbumin, IgG) at 3 ⁇ g/ml in PBS overnight at 4° C. The plates were washed with PBS, and blocked with Blocking Buffer (PBS containing 0.5% BSA) for 2 h at room temperature. Overnight cultures were cleared of E.
- BSA Blocking Buffer
- Binding Buffer Binding Buffer containing 0.05% Tween 20
- PBST PBS containing 0.05% Tween 20
- the plates were incubated with shaking for 2 h at room temperature.
- anti-M13-HRP Pharmacia
- PBS PBS containing 0.05% Tween 20
- a substrate solution containing 0.4 mg/ml ABTS and 0.001% H 2 O 2 in 50 mM phosphate-citrate buffer was added to the wells, and allowed to develop for 40 min after which the plates were read in a plate reader at 405 nm.
- ELISA readings allowed the determination of clone specificity, and antigen-specific clones were sequenced commercially via established methods.
- TrkA-specific binding modules K T W D C R N S G H C V I T F K sNG0035S3.074 A T W D C R D H N F S C V R L S sNG0035S3.089
- Anti-EpCAM peptides were isolated from random peptide libraries that were generated according to Scholle, et al. [Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51]
- the na ⁇ ve peptide libraries displayed cysteine-constrained peptides with cysteines spaced by 4 to 10 random residues.
- EpCam1 EpCAM specific peptide ligands
- EpCam1 peptide ligands were then softly randomized (except cysteine positions) with codons encoding 3-9 residues and moved into a phagemid vector. Phagemid libraries were subsequently affinity selected against EpCAM to isolate peptide ligands optimized for binding (Table X, EpCam2). EpCam2 ligands contain the conserved CXXXXC cysteine spacing. In addition, the majority of anti-EpCam sequences do not contain a lysine residue, which allows for conjugation to free amine groups outside of the binding sequences. Furthermore, anti-EpCam peptide ligands can be genetically fused to URP sequences (of any length) and multimerized using iterative dimerization.
- the resulting anti-EpCAM MURPs can be used to specifically target EpCAM with increased affinity over monomer sequences.
- An example of a tetramer EpCAM-URP amino acid sequence is shown in FIG. 31 . This sequence contains only two lysine residues that are located in the N-terminal Flag-tag. The side chains of these lysine residues are particularly suitable for drug conjugation.
- Binding modules can be affinity matured, or lengthened, by the addition of URP-like linkers and random sequence to the N-terminus, C-terminus, or both N- and C-terminus of the binding sequence.
- FIG. 32 shows the addition of na ⁇ ve cysteine-constrained sequences to an anti-EpCAM binding module.
- Libraries of random sequence additions can be generated using a single-stranded or double-stranded DNA cloning approaches. Once generated, libraries can be affinity selected against the initial target protein or a second protein. For example, an addition library that contains an anti-EpCAM binding module can be used to select sequences that contain 2 or more binding sites to the target protein.
- oligonucleotides were designed to construct a library based on the VEGF-binding 1SS peptide FTCTNHWCPS.
- the oligonucleotides incorporate variations in cysteine distance patterns of the flanking sequences while the VEGF-binding peptide sequence was kept fixed.
- Mixture 1 (from 100 ⁇ M stocks): 100 ⁇ l 70-6, 33 ⁇ l 70-5, 11 ⁇ l 70-4, 3.66 ⁇ l 70-3, 1.2 ⁇ l 70-2, 0.4 ⁇ l 70-1.
- Mixture 2 (from 100 ⁇ M stocks): 100 ⁇ l 70-6R, 33 ⁇ l 70-5R, 11 ⁇ l 70-4R, 3.66 ⁇ l 70-3R, 1.2 ⁇ l 70-2R, 0.4 ⁇ l 70-1R
- Primers 10.0 ⁇ l Assembled mixture, 10.0 ⁇ l 10 ⁇ buffer, 2.0 dNTPs (10 mM), 10.0 ⁇ l LIBPTF (5 ⁇ M), 10.0 ⁇ l LIBPTR (5 ⁇ M), 1.0 ⁇ l cDNA polymerase (Clonetech), 57 ⁇ l DS H 2 0.
- PCR program 95° C. 1 min, (95° C. 15 sec, 54° C. 30 sec, 68° C. 15 sec) ⁇ 25, 68° C. 1 min.
- the product was purified by Amicon column Y10.
- the assembled product was digested with SfiI and BstXI and ligated into the phagemid vector pMP003. Ligation was performed over night at 16° C. in a MJ PCR machine. Ligation then was purified by EtOH precipitation. Transformation into fresh competent ER2738 cells by Electroporation.
- the resulting library was panned against VEGF as described below. Several isolates were identified that showed improved binding to VEGF relative to the 1SS starting sequence. Binding and expression data are shown in FIG. 38 . Sequences and results of Western analysis of buildup clones is shown in FIG. 39 .
- step 13 Continue from step 3) for the next round or store the phage preparation at 4° C.
- Sodium azide may be added to 0.02% (w/v) for long-term storage. Only freshly prepared phage should be used for each round.
- Second round coat 2 wells per library to be screened. Coat the well of a Costar 96-well ELISA plate with 0.25 ⁇ g of VEGF 121 antigen in 25 ⁇ l of PBS. Cover the plate with a plate sealer. Coating can be performed overnight at 4° C. or for 1 h at 37° C.
- binding buffer (blocking buffer+0.5% Tween 20). Aliquot 135 ⁇ l binding buffer per well in low protein-binding 96 well plate.
- Two libraries were designed based on the Plexin scaffold.
- the Pfam protein database was used for phylogenetic alignment of naturally occurring plexin domains as shown in FIG. 35 .
- the middle part of plexin scaffold (Cys24-Gly25-Trp26-Cys27) is conserved in both library designs and served as a crossover region for N- and C-library generation.
- the randomization schemes of both plexin libraries are shown in FIG. 36 .
- the two libraries were generated by overlapping two library-encoding oligos at the crossover region and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) and cloning into phagemid vector pMP003.
- the resulting plexin libraries were designated LMP031 (N terminal library) and LMP032 (C terminal library) and each was represented by a complexity of approximately 5 ⁇ 10 8 independent transformants.
- LMP031 N terminal library
- LMP032 C terminal library
- approximately 24 Carb-resistant clones from each unselected library were analyzed by PCR. Clones that gave a correct size fragment (375 bp) were further analyzed by DNA sequencing. Correct full-length plexin sequences were obtained for 50% and 67% of clones derived from LMP031 and LMP032 libraries, respectively.
- the two libraries were mixed together at 50/50 ratio and panned in parallel against VEGF, death receptor Dr4, ErbB2, and HGFR immobilized on 96-well ELISA plates.
- Four rounds of panning were carried out using 1000 ng of protein target in the first round, 500 ng in the second round, 250 ng in the third round, and 100 ng in the fourth round.
- 192 Carb-resistant clones from each selection were analyzed for binding to 100 ng immobilized protein target, human IgG, Ovalbumin, and BSA by phage ELISA using polyclonal anti-M13 Ab conjugated to horseradish peroxidase for detection.
- target-specific binders are first subcloned into protein expression vector pVS001, then produced as soluble microproteins, and finally purified by heat lysis.
- the purified target-specific microproteins are analysed by protein ELISA to confirm the target recognition, by SDS-PAGE to confirm monomer formation, and by surface plasmon resonance to measure their affinities to target.
- the best clones are used in the next round of library generation to further improve their properties.
- Phage displayed libraries of 10e8 to 10e10 of 3 finger toxin (3FT) scaffolds with partially randomized amino acids of fingertip 1 and descending part of finger 2 or fingertip 3 and ascending part of finger 2 were created by standard methods.
- Three 3FT scaffolds were used as a template for 3FT library generation (fingers 1 and 2 configuration).
- the structure of a 3FT scaffold and a multiple sequence alignment of related sequences is shown in FIG. 33 .
- a library was designed such that two surface loops of the toxin are randomized as illustrated in FIG. 34 .
- the library of partially randomized 3FT scaffold was generated by overlapping four library-encoding oligos at the annealing regions and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) into phagemid vector pMP003.
- the resulting 3FT library was designated LMP041.
- the aim here is to use the peptides that have been identified to be specific for target of interest in order to generate 3SSplus target-specific binders.
- This strategy is illustrated by using VEGF-specific peptide transfer into fingertip 1 of 3FT scaffold and by modifying the AA residues of finger 2, which are in close proximity from target specific sequence to generate high affinity VEGF binders.
- Phage displayed libraries of 10e8 to 10e10 of 3 finger toxin (3FT) scaffolds with VEGF specific sequence of fingertip 1 and partially randomized descending part of finger 2 was created by standard methods as described in example above except 2 random finger 1 forward primers were replaced by F1-VEGF-specific forward primer encoding the following sequence: P S G P S C H T T N H W P I S A V T C P P.
- the focused (VEGF-specific) 3FT scaffold library with partially randomized finger 2 was generated by overlapping four library-encoding oligos at the annealing regions and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) into phagemid vector pMP003.
- the resulting 3FT library was designated LMP042.
- the plasma half-life of MURPs can be measured after i.v. or i.p. injection of the MURP into catheterized rats essentially as described by [Pepinsky, R. B., et al. (2001) J Pharmacol Exp Ther, 297: 1059-66]. Blood samples can be withdrawn at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 5 h, 1 d, 2 d, 3 d) and the plasma concentration of the MURP can be measured using ELISA. Pharmacokinetic parameters can be calculated using WinNonlin version 2.0 (Scientific Consulting Inc., Apex, N.C.). To analyze the effect of the URP module one can compare on plasma half-life of a protein containing the URP module with the plasma half-life of the same protein lacking the URP module.
- Solubility of MURPs can be determined by concentrating purified samples of MURPs in physiological buffers like phosphate buffered saline to various concentrations in the range of 0.01 mg/ml to 10 mg/ml. Samples can be incubated for up to several weeks. Samples where the concentration exceeds the solubility of the MURP show precipitation as indicated by turbidity, which can be measured in an absorbance reader. On can remove precipitated material by centrifugation or filtration and measure the concentration of remaining protein in the supernatant using a protein assay like the Bradford assay of by measuring the absorbance at 280 nm. Solubility studies can be accelerated by freezing the samples at ⁇ 20 C and subsequent thawing. This process frequently leads to the precipitation of poorly soluble proteins.
- MURPs of interest into microtiter plates and control proteins in other wells of the plate. Subsequently, one can add serum samples of interest to the wells for 1 hour. Subsequently, the wells can be washed with a plate washer. Bound serum proteins can be detected by adding antibodies against serum proteins that have been conjugated with enzymes like horse radish peroxidase or alkaline phosphatase for detection. Another way to detec serum binding to MURPs to add the MURP of interest to serum for about 1 hour to allow binding. Subsequently, one can immunoprecipitate the MURP using an antibody against an epitope in the MURP sequence. The precipitated samples can be analyzed by PAGE and optionally by Western to detect any proteins that co-precipitated with the MURP. One can identify the serum proteins that show co-precipitation by mass spectrometry.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Pathology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Crystallography & Structural Chemistry (AREA)
- General Physics & Mathematics (AREA)
Abstract
Description
- This application claims the benefit of U.S. Provisional Application No. 60/743,410 filed Mar. 6, 2006, which application is incorporated herein by reference. This application is a continuation-in-part application of Ser. Nos. 11/528,927 and 11/528,950, filed on Sep. 27, 2006, which in turn claim priority to provisional applications Ser. Nos. 60/721,270, 60/721,188, filed on Sep. 27, 2005 and 60/743,622 filed on Mar. 21, 2006, all of which are herein incorporated by reference in their entirety.
- It has been well documented that properties of proteins, in particular plasma clearance and immunogenicity, can be improved by attaching hydrophilic polymers to these proteins (Kochendoerfer, G. (2003) Expert Opin Biol Ther, 3: 1253-61), (Greenwald, R. B., et al. (2003) Adv Drug Deliv Rev, 55: 217-50), (Harris, J. M., et al. (2003) Nat Rev Drug Discov, 2: 214-21). Examples of polymer-modified proteins that have been approved by the FDA for treatment of patients are Adagen, Oncaspar, PEG-Intron, Pegasys, Somavert, and Neulasta. Many more polymer-modified proteins are in clinical trials. These polymers exert their effect by increasing the hydrodynamic radius (also called Stokes' radius) of the modified protein relative to the unmodified protein, which reduces the rate of clearance by kidney filtration (Yang, K., et al. (2003) Protein Eng, 16: 761-70). In addition, polymer attachment can reduce interaction of the modified protein with other proteins, cells, or surfaces. In particular, polymer attachment can reduce interactions between the modified protein and antibodies and other components of the immune system thus reducing the formation of a host immune response to the modified protein. Of particular interest is protein modification by PEGylation, i.e. by attaching linear or branched polymers of polyethylene glycol. Reduced immunogenicity upon PEGylation was shown for example for phenylalanine ammonia lyase (Gamez, A., et al. (2005) Mol Ther, 11: 986-9), antibodies (Deckert, P. M., et al. (2000) Int J Cancer, 87: 382-90.), Staphylokinase (Collen, D., et al. (2000) Circulation, 102: 1766-72), and hemoglobin (Jin, C., et al. (2004) Protein Pept Lett, 11: 353-60). Typically, such polymers are conjugated with the protein of interest via a chemical modification step after the unmodified protein has been purified.
- Various polymers can be attached to proteins. Of particular interest are hydrophilic polymers that have flexible conformations and are well hydrated in aqueous solutions. A frequently used polymer is polyethylene glycol (PEG). These polymers tend to have large hydrodynamic radi relative to their molecular weight (Kubetzko, S., et al. (2005) Mol Pharmacol, 68: 1439-54). The attached polymers tend to have limited interactions with the protein they have been attached to and thus the polymer-modified protein retains its relevant functions.
- The chemical conjugation of polymers to proteins requires complex multi-step processes. Typically, the protein component needs to be produced and purified prior to the chemical conjugation step. The conjugation step can result in the formation of product mixtures that need to be separated leading to significant product loss. Alternatively, such mixtures can be used as the final pharmaceutical product. Some examples are currently marketed PEGylated Interferon-alpha products that are used as mixtures (Wang, B. L., et al. (1998) J Submicrosc Cytol Pathol, 30: 503-9; Dhalluin, C., et al. (2005) Bioconjug Chem, 16: 504-17). Such mixtures are difficult to manufacture and characterize and they contain isomers with reduced or no therapeutic activity.
- Methods have been described that allow the site-specific addition of polymers like PEG. Examples are the selective PEGylation at a unique glycosylation site of the target protein or the selective PEGylation of a non-natural amino acid that has been engineered into the target proteins. In some cases it has been possible to selectively PEGylate the N-terminus of a protein while avoiding PEGylation of lysine side chains in the target protein by carefully controlling the reaction conditions. Yet another approach for the site-specific PEGylation of target proteins is the introduction of cysteine residues that allow selective conjugation. All these methods have significant limitations. The selective PEGylation of the N-terminus requires careful process control and side reactions are difficult to eliminate. The introduction of cysteines for PEGylation can interfere with protein production and/or purification. The specific introduction of non-natural amino acids requires specific host organisms for protein production. A further limitation of PEGylation is that PEG is typically manufactured as a mixture of polymers with similar but not uniform length. The same limitations are inherent in many other chemical polymers.
- Chemical conjugation using multifunctional polymers which would allow the synthesis of products with multiple protein modules is even more complex then the polymer conjugation of a single protein domain.
- Recently, it has been observed that some proteins of pathogenic organisms contain repetitive peptide sequences that seem to lead to a relatively long serum halflife of the proteins containing these sequences (Alvarez, P., et al. (2004) J Biol Chem, 279: 3375-81). It has also been demonstrated that oligomeric sequences that are based on such pathogen-derived repetitive sequences can be fused to other proteins resulting in increased serum halflife. However, these pathogen-derived oligomers have a number of deficiencies. The pathogen-derived sequences tend to be immunogenic. It has been described that the sequences can be modified to reduce their immunogenicity. However, no attempts have been reported to remove T cell epitopes from the sequences contributing to the formation of immune reactions. Furthermore, the pathogen-derived sequences have not been optimized for pharmacological applications which require sequences with good solubility and a very low affinity for other target proteins.
- Thus there is a significant need for compositions and methods that would allow one to combine multiple polymer modules and multiple protein modules into defined multidomain products.
- The present invention provides an unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm. In a related embodiment, the present invention provides an unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP has an in vitro serum degradation half-life greater than about 24 hours, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm. The subject URP can comprises a non-natural amino acid sequence. Where desired, the URP is selected for incorporation into a heterologous protein, and wherein upon incorporation the URP into a heterologous protein, said heterologous protein exhibits a longer serum secretion half-life and/or higher solubility as compared to the corresponding protein that is deficient in said URP. The half-life can be extended by two folds, three folds, five folds, ten folds or more. In some aspects, incorporation of the URP into a heterologous protein results in at least a 2-fold, 3-fold, 4-fold, 5-fold or more increase in apparent molecular weight of the protein as approximated by size exclusion chromatography. In some aspects, the URPs has a Tepitope score less than −3.5 (e.g., −4 or less, −5 or less). In some aspects, the URPs can contain predominantly hydrophilic residues. Where desired, at least 50% of the amino acids of the URP are devoid of secondary structure as determined by Chou-Fasman algorithm. The glycine residues contained in the URP may constitute at least about 50% of the total amino acids of the URP. In some aspect, any one type of the amino acids alone selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) contained in the URP constitutes more than about 20%, 30%, 40%, 50%, 60% or more of the total amino acids of the URP. In some aspects, the URP comprises more than about 100, 150, 200 or more contiguous amino acids.
- The present invention also provides a protein comprising one or more of the subject URPs, wherein the subject URPs are heterologous with respect to the protein. The total length of URPs in aggregation can exceed about 40, 50, 60, 100, 150, 200, or more amino acids. The protein can comprise one or more functional modules selected from the group consisting of effector module, binding module, N-terminal module, C-terminal module, and any combinations thereof. Where desired, the subject protein comprises a plurality of binding modules, wherein the individual binding modules exhibit binding specificities to the same or different targets. The binding module may comprise a disulfide-containing scaffold formed by intra-scaffold pairing of cysteines. The binding module may bind to a target molecule target is selected from the group consisting of cell surface protein, secreted protein, cytosolic protein, and nuclear protein. The target can be an ion channel and/or GPCR. Where desired, the effector module can be a toxin. The subject URP-containing protein typically an extended serum secretion half-life by at least 2, 3, 4, 5, 10 or more folds as compared to a corresponding protein that is deficient in said URP.
- In a seperate embodiment, the present invention provides a non-naturally occurring protein comprising at least 3 repeating units of amino acid sequences, each of the repeating unit comprising at least 6 amino acids, wherein the majority of segments comprising about 6 to about 15 contiguous amino acids of the at least 3 repeating units are present in one or more native human proteins. In one aspect, the majority of the segments, or each segment comprising about 9 to about 15 contiguous amino acids within the repeating units are present in one or more native human proteins. The segments can comprise about 9 to about 15 amino acids. The three repeating units may share substantial sequence homology, e.g., share sequence identify of greater than about 50%, 60%, 70%, 80%, 90% or 100% when aligned. Such non-natural protein may also comprise one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules. Where desired, the non-natural protein may comprise individual repeating unit having the subject unstructured recombinant polymer (URP).
- The present invention also provides recombinant polynucleotides comprising coding sequences that encode the subject URPs, URP-containing proteins, microproteins and toxins. Also provided in the present invention are vectors containing the subject polynucleotides, host cells harboring the vectors, genetic packages displaying the subject URPs, URP-containing proteins, toxins and any other proteinaceous entities disclosed herein. Further provided are selectable library of expression vectors of the present invention.
- The present invention also provides method of producing a protein comprising an unstructured recombinant polymer (URP). The method involves (i) providing a host cell comprising a recombinant polynucleotide encoding the protein, said protein comprising one or more URP, said URP comprising at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm; and (ii) culturing said host cell in a suitable culture medium under conditions to effect expression of said protein from said polynucleotide. Suitable host cells are eukaryotic (e.g., CHO cells) and prokaryotic cells.
- The present invention also provides a method of increasing-serum secretion half-life of a protein, comprising: fusing said protein with one or more unstructured recombinant polymers (URPs), wherein the URP comprises at least about 40 contiguous amino acids, and wherein (a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP; and/or (b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm; and wherein said URP is substantially incapable of non-specific binding to a serum protein.
- Also provided in the present invention is a method of detecting the presence or absence of a specific interaction between a target and an exogenous protein that is displayed on a genetic package, wherein said protein comprises one or more unstructured recombinant polymer (URP), the method comprising: (a) providing a genetic package displaying a protein that comprises one or more unstructured recombinant polymers (URPs); (b) contacting the genetic package with the target under conditions suitable to produce a stable protein-target complex; and (c) detecting the formation of the stable protein-target complex on the genetic package, thereby detecting the presence of a specific interaction. The method may further comprises obtaining a nucleotide sequence from the genetic package that encodes the exogenous protein. In some aspects, the presence or absence of a specific interaction is between the URP and a target comprising a serum protein. In some aspects, the presence or absence of a specific interaction is between the URP and a target comprising a serum protease.
- Further included in the present invention is a genetic package displaying a microprotein, wherein said microprotein retains binding capability to its native target. In some aspects, the microprotein exhibits binding capability towards at least one family of ion channel selected from the group consisting of a sodium, a potassium, a calcium, an acetylcholine, and a chlorine channel. Where desired, the microprotein is an ion-channel-binding microprotein, and is modified such that (a) the microprotein binds to a different family of channel as compared to the corresponding unmodified microprotein; (b) the microprotein binds to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein; (c) the microprotein binds to a different species of the same subfamily of channel as compared to the corresponding unmodified microprotein; (d) the microprotein binds to a different site on the same channel as compared to the corresponding unmodified microprotein; and/or (e) the microprotein binds to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein. In some aspect, the microprotein is a toxin. The present invention also provides a library of genetic packages displaying the subject microproteins and/or toxins. Where desired, the genetic package displays a proteinaceous toxin that retains in part or in whole its toxicity spectrum. The toxin can be derived from a single toxin protein, or derived from a family of toxins. The present invention also provides a library of genetic packages wherein the library displays a family of toxins, wherein the family retains in part or in whole its native toxicity spectrum.
- The present invention further provides a protein comprising a plurality of ion-channel binding domains, wherein individual domains are microprotein domains that have been modified such that (a) the microprotein domains bind to a different family of channel as compared to the corresponding unmodified microprotein domains; (b) the microprotein domains bind to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein domains; (c) the microprotein domains bind to a different species of the same subfamily as compared to the corresponding unmodified microprotein domains; (d) the microprotein domains bind to a different site on the same channel as compared to the corresponding unmodified microprotein domains; (e) the microprotein domains bind to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein domains; and/or (f) the microprotein domains bind to the same site of the same channel and yield the same biological effect as compared to the corresponding unmodified microprotein domains.
- Also embodied in the invention is a method of obtaining a microprotein with desired property, comprising: (a) providing a subject library; and (b) screening the selectable library to obtain at least one phage displaying a microprotein with the desired property. Polynucleotides, vectors, genetic packages, host cells for use in any one of the disclosed methods are also provided.
- All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
- The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
-
FIG. 1 shows the modular components of an MURP. Binding modules, effector modules, and multimerization modules are depicted as circles. URP modules, N-terminal, and C-terminal modules are shown as rectangles. -
FIG. 2 shows examples of modular architectures of MURPs. Binding modules (BM) in one MURP can have identical or differing target specificities. -
FIG. 3 shows that a repeat protein that is based on a human sequence can contain novel amino acid sequences, which can contain T cell epitopes. These novel sequences are formed at the junction between neighboring repeat units. -
FIG. 4 illustrates the design of a URP sequence that is a repeat protein based on three human donor sequences D1, D2, and D3. The repeating unit of this URP was chosen such that even 9-mer sequences that span the junction between neighboring units can be found in at least one of the human donor sequences. -
FIG. 5 Example of a URP sequences that is a repeat protein based on the sequences of three human proteins. The lower portion of the figure illustrates that all 9-mer subsequences in the URP occur in at least one of the human donor proteins. -
FIG. 6 Example based URP sequence based on the human POU domain residues 146-182. -
FIG. 7 shows the advantage of separating modules with information rich sequences by inserting URP modules between such sequences. The left side of the figure shows that the direct fusion of modules A and B leads to novel sequences in the junction region. These junction sequences can be epitopes. The right half of the figure shows that the insertion of a URP module between module A and B prevents the formation of such junction sequences that contain partial sequences from modules A and B. Instead, the termini of modules A and B yield junction sequences that contain URP sequences and thus are predicted to have low immunogenicity. -
FIG. 8 shows drug delivery constructs that are based on URPs. The drug molecules depicted as hexagons are chemically conjugated to the MURP. -
FIG. 9 shows and MURP containing a protease-sensitive site. The URP module is designed such that it blocks the effector module from its function. Protease cleavage removes a portion of the URP module and results in increased activity of the effector function. -
FIG. 10 shows how an URP module can act as a linker between a binding module and an effector module. The binding module can bind to a target and as a consequence it increases the local concentration of the effector module in the proximity of the target. -
FIG. 11 Shows a process to construct genes encoding URP sequences from libraries of short URP modules. The URP module library can be inserted into a stuffer vector that contains green fluorescent protein (GFP) as a reporter to facilitate the identification of URP sequences with high expression. The figure illustrates that genes encoding long URP sequences can be build by iterative dimerization. -
FIG. 12 shows MURPs that contain multiple binding modules for death receptors. Death receptors are triggered by trimerization and thus MURPs containing at least three binding elements for one death receptor particularly potent in inducing cell death. The lower portion of the figure illustrates that one can increase the specificity of the MURP for diseased tissue by adding one or more binding modules with specificity for tumor tissue. -
FIG. 13 shows a MURP that comprises four binding modules (rectangles) with specificity for a tumor antigen with an effector module likeinterleukin 2. -
FIG. 14 shows the flow chart for the construction of URP modules with 288 residues. The URP modules were constructed as fusion proteins with GFP. Libraries of URP modules with 36 amino acids were constructed first followed by iterative dimerization to yield URP modules with 288 amino acids (rPEG_H288 and rPEG_J288). -
FIG. 15 Amino acid and nucleotide sequence of a URP module with 288 amino acids (rPEG_J288). -
FIG. 16 Amino acid and nucleotide sequence of a URP module with 288 amino acids (rPEG_H288). -
FIG. 17 Amino acid sequence of a serine-rich sequence region of the human protein dentin sialophosphoprotein. -
FIG. 18 shows a depot derivative of a MURP. The protein contains two cysteine residues that can form a weak SS bridge. The protein can be manufactured with the SS bridge intact. It can be formulated and injected into patients in reduced form. After injection it will be oxidized in proximity to the injection site and as a result in can form a high molecular weight polymer with very limited diffusivity. The active MURP can slowly leach from the injection site by limited proteolysis or limited reduction of the cross linking SS bond. -
FIG. 19 shows a depot form of a MURP. The MURP has very limited diffusivity at the injection site and can be liberated from the injection site by limited proteolysis. -
FIG. 20 shows a depot form of a MURP that contains a histidine-rich sequence. The MURP can be formulated and injected in combination with insoluble beads that contain immobilized nickel. The MURP binds to the nickel beads at the injection site and is released slowly into the circulation. -
FIG. 21 shows MURPs that contain multimerization modules. The upper part of the figure shows an MURP that contains one dimerization sequence. As a result it forms a dimer which effectively doubles its molecular weight. The center of the figure shows three MURP designs that comprise two multimerization sequences. Such MURPs can form multimers with very high effective molecular weight. The lower part of the figure illustrated an MURP that contains multiple RGD sequences that are known to bind to cell surface receptors and thus confer half-life. -
FIG. 22 Shows a variety of MURPs that are designed to block or modulate ion channel function. Circles indicate binding modules with specificity for ion channels. These binding modules can be derived or identical to natural toxins with affinity for ion channel receptors. The figure illustrates that other binding domains can be added on either side of the ion channel-specific binding modules thus conferring the MURPs increased efficacy or specificity for a particular cell type. -
FIG. 23 shows several MURP designs for increased half-life. Increased effective molecular weight can be achieved by increasing chain length (A), chemical multimerization (B), adding multiple copies of binding modules into a molecule separated by non-binding sites (C), construction of chemical multimers similar to C (D, E), including multimerization sequences (F). -
FIG. 24 shows MURPs that can be formed by chemical conjugation of binding modules to a recombinant URP sequence. The URP sequence is designed to contain multiple lysine residues (K) as conjugation sites. -
FIG. 25 shows the design of a library of 2SS binding modules. The sequences contain a constant 1SS sequence in the center which is flanked by random sequences that contain cysteine residues in varying distance from the 1SS core. -
FIG. 26 shows the design of a library of 2SS binding modules. The sequences contain a constant 1SS sequence in the center which is flanked by random sequences that contain cysteine residues in varying distance from the 1SS core. -
FIG. 27 shows the design of a library of dimers of 1SS binding modules. Initially, a collection of 1SS binding modules is amplified by two PCR reactions. The resulting PCR products are combined and dimers are generated in a subsequent PCR step. -
FIG. 28 show the Western analysis of a fusion protein containing the 288 amino acid URP sequence rPEG_J288 after incubation of up to 3 days in 50% mouse serum. -
FIG. 29 shows results of a binding assay testing for pre-existing antibodies against a URP sequence of 288 amino acids. -
FIG. 30 shows the binding of MURPs containing one (Monomer), two (Dimer), four (Tetramer), or zero (rPEG36) binding modules with specificity for VEGF which was coated to microtiter plates. -
FIG. 31 shows the amino acid sequence of an MURP with specificity for EpCAM. The sequence contains four binding modules with affinity for EpCAM (underlined). The sequence contains an N-terminal Flag sequence which contains the only two lysine residues of the entire sequence. -
FIG. 32 shows the design of 1SS addition libraries. Random 1SS modules can be added to the N- or C-terminus of a pre-selected binding module or simultaneously to both sides. -
FIG. 33 shows the alignment of three finger toxin-related sequences. The figure also shows a 3D structure that was solved by NMR. -
FIG. 34 shows the design of a three-finger toxin-based library. Residues designated X were randomized. The codon choice for each random position is indicated. -
FIG. 35 shows the alignment of plexin-related sequences. -
FIG. 36 shows the design of a plexin-based library. Residues designated X were randomized. The codon choice for each random position is indicated. -
FIG. 37 Sequences of plexin-related binding modules with specificity for DR4, ErbB2, and HGFR. -
FIG. 38 shows a binding assay for microprotein-based binding domains with specificity for VEGF. -
FIG. 39 shows sequences of 2SS and 3SS binding modules that were isolated from buildup libraries with specificity for VEGF. The upper part of the protein shows PAGE gel analysis of the proteins purified by heat-lysis. -
FIG. 40 shows cloning steps to construct the URP sequence rPEG_J72. -
FIG. 41 shows the construction of a library of URP modules with 36 amino acids called rPEG_J36. The region encoding rPEG_J36 was assembled by ligating three shorter segments encoding rPEG_J12 and a stopper module. -
FIG. 42 shows the nucleotide sequence and translation of the stuffer vector pCW0051. The stuffer region is flanked by BsaI and BbsI sites and contains multiple stop codons. -
FIG. 43 shows a PAGE gel of the purification of the URP rPEG_J288 fused to GFP.Lane 2 shows the cell lysate; lane 3: product purified by IMAC; lane 4: product purified by anti-Flag. -
FIG. 44 Amino acid sequence of fusion proteins between rPEG_J288 and human effector domains interferon alpha, G-CSF, and human growth hormone. -
FIG. 45 shows the Western analysis of expression of fusion proteins between rPEG_J288 and human growth hormone (lanes 1 and 2), interferon alpha (lanes 3 and 4), and GFP (lanes 5 and 6). Both soluble and insoluble material was analyzed for each protein. -
FIG. 46 shows the design of MURPs based on the toxin OSK1. The figure shows that URP sequences and/or binding modules can be added to either side of OSK1 -
FIG. 47 depicts exemplary product formats comprising the subject URPs. - While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
- The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
- As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
- The terms “polypeptide”, “peptide”, “amino acid sequence” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
- A “repetitive sequence” refers to an amino acid sequence that can be described as an oligomer of repeating peptide sequences, forming direct repeats, or inverted repeats or alternating repeats of multiple sequence motifs. These repeating oligomer sequences can be identical or homologous to each other, but there can also be multiple repeated motifs. Repetitive sequences are characterized by a very low information content. A repetitive sequence is not a required feature of a URP and in some cases a non-repetitive sequence will in fact be preferred.
- Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M et al. (see Levitt, M (1976) J Mol Biol 104, 59, #3233, which is listed in Hopp, T P, et al. (1981) Proc Natl Acad Sci USA 78, 3824, #3232). Examples of “hydrophilic amino acids” are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of “hydrophobic amino acids” are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
- The term “denatured conformation” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperatures. Peptides in denatured conformation have characteristic CD spectra and they are generally characterized by a lack of long range interactions as determined by e.g., NMR. Denatured conformation and unfolded conformation will be used synonymously.
- The terms “unstructured protein (UNP) sequences” and “unstructured recombinant polymer” (URP) are used herein interchanageably. The terms refer to amino acid sequences that share commonality with denatured peptide sequences, e.g., exhibiting a typical behavior like denatured peptide sequences, under physiological conditions, as detailed herein. URP sequences lack a defined tertiary structure and they have limited or no secondary structure as detected by, e.g., Chou-Fasman algorithm.
- As used herein, the term “cell surface proteins” refers to the plasma membrane components of a cell. It encompasses integral and peripheral membrane proteins, glycoproteins, polysaccharides and lipids that constitute the plasma membrane. An integral membrane protein is a transmembrane protein that extends across the lipid bilayer of the plasma membrane of a cell. A typical integral membrane protein consists of at least one membrane spanning segment that generally comprises hydrophobic amino acid residues. Peripheral membrane proteins do not extend into the hydrophobic interior of the lipid bilayer and they are bound to the membrane surface via covalent or noncovalent interaction directly or indirectly with other membrane components.
- The terms “membrane”, “cytosolic”, “nuclear” and “secreted” as applied to cellular proteins specify the extracellular and/or subcellular location in which the cellular protein is mostly, predominantly, or preferentially localized.
- “Cell surface receptors” represent a subset of membrane proteins, capable of binding to their respective ligands. Cell surface receptors are molecules anchored on or inserted into the cell plasma membrane. They constitute a large family of proteins, glycoproteins, polysaccharides and lipids, which serve not only as structural constituents of the plasma membrane, but also as regulatory elements governing a variety of biological functions.
- The term “module” refers to a portion of a protein that is physically or functionally distinguished from other portions of the protein or peptide. A module can comprise one or more domains. In general, a module or domain can be a single, stable three-dimensional structure, regardless of size. The tertiary structure of a typical domain is stable in solution and remains the same whether such a member is isolated or covalently fused to other domains. A domain generally has a particular tertiary structure formed by the spatial relationships of secondary structure elements, such as beta-sheets, alpha helices, and unstructured loops. In domains of the microprotein family, disulfide bridges are generally the primary elements that determine tertiary structure. In some instances, domains are modules that can confer a specific functional activity, such as avidity (multiple binding sites to the same target), multi-specificity (binding sites for different targets), halflife (using a domain, cyclic peptide or linear peptide) which binds to a serum protein like human serum albumin (HSA) or to IgG (hIgG1, 2, 3 or 4) or to red blood cells. Functionally-defined domains have a distinct biological function(s). The ligand-binding domain of a receptor, for example, is that domain that binds ligand. An antigen-binding domain refers to the part of an antigen-binding unit or an antibody that binds to the antigen. Functionally-defined domains need not be encoded by contiguous amino acid sequences. Functionally-defined domains may contain one or more physically-defined domain. Receptors, for example, are generally divided into the extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain. A “membrane anchorage domain” refers to the portion of a protein that mediates membrane association. Generally, the membrane anchorage domain is composed of hydrophobic amino acid residues. Alternatively, the membrane anchorage domain may contain modified amino acids, e.g. amino acids that are attached to a fatty acid chain, which in turn anchors the protein to a membrane.
- “Non-naturally occurring” as applied to a protein means that the protein contains at least one amino acid that is different from the corresponding wildtype or native protein. Non-natural sequences can be determined by performing BLAST search using, e.g., the lowest smallest sum probability where the comparison window is the length of the sequence of interest (the queried) and when compared to the non-redundant (“nr”) database of Genbank using BLAST 2.0. The BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
- A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.
- As used herein, the term “isolated” means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. As is apparent to those of skill in the art, a non-naturally occurring the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
- “Linked” and “fused” or “fusion” are used interchangeably herein. These terms refer to the joining together of two more chemical elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more open reading frames (OFRs) to form a continuous longer OFR, in a manner that maintains the correct reading frame of the original OFRs. Thus, the resulting recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original OFRs (which segments are not normally so joined in nature.)
- In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide which is known to comprise additional residues in one or both directions.
- “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
- The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
- “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
- The terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
- A “vector” is a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
- The “target” as used in the context of MURPs is a biochemical molecule or structure to which the Binding Module or the URP-linked Binding Module can bind and where the binding event results in a desired biological activity. The target can be a protein ligand or receptor that is inhibited, activated or otherwise acted upon by the t protein. Examples of targets are hormones, cytokines, antibodies or antibody fragments, cell surface receptors, kinases, growth factors and other biochemical structures with biological activity.
- A “functional module” can be any non-URP in a protein product. Thus a functional module can be a binding module (BM), an effector module (EM), a multimerization module (MM), a C-terminal module (CM), or an N-terminal module (NM). In general, functional modules are characterized by a high information content of their amino acid sequence, i.e they contain many different amino acids and many of these amino acids are important for the function of a functional module. A functional module typically has secondary and tertiary structure, may be a folded protein domain and may contain 1, 2, 3, 4, 5 or more disulfide bonds.
- The term ‘microproteins’ refers to a classification in the SCOP database. Microproteins are usually the smallest proteins with a fixed structure and typically but not exclusively have as few as 15 amino acids with two disulfides or up to 200 amino acids with more than ten disulfides. A microprotein may contain one or more microprotein domains. Some microprotein domains or domain families can have multiple more-or-less stable and multiple more or less similar structures which are conferred by different disulfide bonding patterns, so the term stable is used in a relative way to differentiate microproteins from peptides and non-microprotein domains. Most microprotein toxins are composed of a single domain, but the cell-surface receptor microproteins often have multiple domains. Microproteins can be so small because their folding is stabilized either by disulfide bonds and/or by ions such as Calcium, Magnesium, Manganese, Copper, Zinc, Iron or a variety of other multivalent ions, instead of being stabilized by the typical hydrophobic core.
- The term “scaffold” refers to the minimal polypeptide ‘framework’ or ‘sequence motif’ that is used as the conserved, common sequence in the construction of protein libraries. In between the fixed or conserved residues/positions of the scaffold lie variable and hypervariable positions. A large diversity of amino acids is provided in the variable regions between the fixed scaffold residues to provide specific binding to a target molecule. A scaffold is typically defined by the conserved residues that are observed in an alignment of a family of sequence-related proteins. Fixed residues may be required for folding or structure, especially if the functions of the aligned proteins are different. A full description of a microprotein scaffold may include the number, position or spacing and bonding pattern of the cysteines, as well as position and identity of any fixed residues in the loops, including binding sites for ions such as Calcium.
- The “fold” of a microprotein is largely defined by the linkage pattern of the disulfide bonds (i.e., 1-4, 2-6, 3-5). This pattern is a topological constant and is generally not amenable to conversion into another pattern without unlinking and relinking the disulfides such as by reduction and oxidation (redox agents). In general, natural proteins with related sequences adopt the same disulfide bonding patterns. The major determinants are the cysteine distance pattern (CDP) and some fixed non-cys residues, as well as a metal-binding site, if present. In few cases the folding of proteins is also influenced by the surrounding sequences (ie pro-peptides) and in some cases by chemical derivatization (ie gamma-carboxylation) of residues that allow the protein to bind divalent metal ions (ie Ca++) which assists their folding. For the vast majority of microproteins such folding help is not required.
- However, proteins with the same bonding pattern may still comprise multiple folds, based on differences in the length and composition of the loops that are large enough to give the protein a rather different structure. An example are the conotoxin, cyclotoxin and anato domain families, which have the same DBP but a very different CDP and are considered to be different folds. Determinants of a protein fold are any attributes that greatly alter structure relative to a different fold, such as the number and bonding pattern of the cysteines, the spacing of the cysteines, differences in the sequence motifs of the inter-cysteine loops (especially fixed loop residues which are likely to be needed for folding, or in the location or composition of the calcium (or other metal or co-factor) binding site.
- The term “disulfide bonding pattern” or “DBP” refers to the linking pattern of the cysteines, which are numbered 1-n from the N-terminus to the C-terminus of the protein. Disulfide bonding patterns are topologically constant, meaning they can only be changed by unlinking one or more disulfides such as using redox conditions. The possible 2-, 3-, and 4-disulfide bonding patterns are listed below in paragraphs 0048-0075.
- The term “cysteine distance pattern” or “CDP” refers to the number of non-cysteine amino acids that separate the cysteines on a linear protein chain. Several notations are used: C5C0C3C equals C5CC3C equals CxxxxxCCxxxC.
- The term ‘Position n6’ or ‘n7=4’ refers to the intercysteine loops and ‘n6’ is defined as the loop between C6 and C7; ‘n7=4’ means the loop between C7 and C8 is 4 amino acids long, not counting the cysteines.
- Serum degradation resistance—Proteins can be eliminated by degradation in the blood, which typically involves proteases in the serum or plasma. The serum degradation resistance is measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (ie 0.25, 0.5, 1, 2, 4, 8, 16 days) at 37 C. The samples for these timepoints are then run on a western assay and the protein is detected with an antibody. The antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred. The timepoint where 50% of the protein is degraded, as judged by western, is the serum degradation halflife of the protein.
- Serum protein binding—While the MURP typically has a number of modules that bind to cell-surface targets and/or serum proteins, it is desirable that the URP substantially lack unintended activities. The URP should be designed to minimize avoid interaction with (binding to) serum proteins, including antibodies. Different URP designs can be screened for serum protein binding by ELISA, immobilizing the serum proteins and then adding the URP, incubating, washing and then detecting the amount of bound URP. One approach is to detect the URP using an antibody that recognizes a tag that has been added to the URP. A different approach is to immobilize the URP (such as via a fusion to GFP) and come in with human serum, incubating, washing, and then detecting the amount of human antibodies that remain bound to the URP using secondary antibodies like goat anti-human IgG. Using these approaches we have designed our URPs to show very low levels of binding to serum proteins. However, in some applications binding to serum proteins or serum-exposed proteins is desired, for example because it can further extend the secretion halflife. In such cases one can use these same assays to design URPs that bind to serum proteins or serum-exposed proteins such as HSA or IgG. In other cases the MURP can be given binding modules that contain peptides that have been designed to bind to serum proteins or serum-exposed proteins such as HAS or IgG.
- One aspect of the present invention is the design of unstructured recombinant polymers (URPs). The subject URPs are particularly useful for generating recombinant proteins of therapeutic and/or diagnostic value. The subject URPs exhibit one or more following features.
- The subject URPs comprise amino acid sequences that typically share commonality with denatured peptide sequences under physiological conditions. URP sequences typically behave like denatured peptide sequences under physiological conditions. URP sequences lack well defined secondary and tertiary structures under physiological conditions. A variety of methods have been established in the art to ascertain the second and tertiary structures of a given polypeptide. For example, the secondary structure of a polypeptide can be determined by CD spectroscopy in the “far-UV” spectral region (190-250 nm). Alpha-helix, beta-sheet, and random coil structures each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be ascertained via certain computer programs or algorithms such as the Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45). For a given URP sequence, the algorithm can predict whether there exists some or no secondary structure at all. In general, URP sequences will have spectra that resemble denatured sequences due to their low degree of secondary and tertiary structure. Where desired, URP sequences can be designed to have predominantly denatured conformations under physiological conditions. URP sequences typically have a high degree of conformational flexibility under physiological conditions and they tend to have large hydrodynamic radii (Stokes' radius) compared to globularproteins of similar molecular weight. As used herein, physiological conditions refer to a set of conditions including temperature, salt concentration, pH that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and at adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers is listed in Sambrook et al. (1989) supra and hence is not detailed herein. Physiologically relevant temperature ranges from about 25° C. to about 38° C., and preferably from about 30° C. to about 37° C.
- The subject URPs can be sequences with low immunogenicity. Low immunogenicity can be a direct result of the conformational flexibility of URP sequences. Many antibodies recognize so-called conformational epitopes in protein antigens. Conformational epitopes are formed by regions of the protein surface that are composed of multiple discontinuous amino acid sequences of the protein antigen. The precise folding of the protein brings these sequences into a well-defined special configuration that can be recognized by antibodies. Preferred URPs are designed to avoid formation of conformational epitopes. For example, of particular interest are URP sequences having a low tendency to adapt compactly folded conformations in aqueous solution. In particular, low immunogenicity can be achieved by choosing sequences that resist antigen processing in antigen presenting cells, choosing sequences that do not bind MHC well and/or by choosing sequences that are derived from human sequences.
- The subject URPs can be sequences with a high degree of protease resistance. Protease resistance can also be a result of the conformational flexibility of URP sequences. Protease resistance can be designed by avoiding known protease recognition sites. Alternatively, protease resistant sequences can be selected by phage display or related techniques from random or semi-random sequence libraries. Where desired for special applications, such as slow release from a depot protein, serum protease cleavage sites can be built into an URP. Of particular interest are URP sequences with high stability (e.g., long serum half-life, less prone to cleavage by proteases present in bodily fluid) in blood.
- The subject URP can also be characterized by the effect in that wherein upon incorporation of it into a protein, the protein exhibits a longer serum half-life and/or higher solubility as compared to the corresponding protein that is deficient in the URP. [Methods of ascertaining serum half-life are known in the art (see e.g., Alvarez, P., et al. (2004) J Biol Chem, 279: 3375-81). One can readily determine whether the resulting protein has a longer serum half-life as compared to the unmodified protein by praciting any methods available in the art or exemplified herein.
- The subject URP can be of any length necessary to effect (a) extension of serum half-life of a protein comprising the URP; (b) an increase in solubility of the resulting protein; (c) an increased resistance to protease; and/or (d) a reduced immunogenicity of the resulting protein that comprises the URP. Typically, the subject URP has about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or more contiguous amino acids. When incorporated into a protein, the URP can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs. Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 40 amino acids. Preferably, the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- URPs may have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 or even 13.0.
- In general, URP sequences are rich in hydrophilic amino acids and contain a low percentage of hydrophobic or aromatic amino acids. Suitable hydrophilic residues include but are not limited to glycine, serine, aspartate, glutamate, lysine, arginine, and threonine. Hydrophobic residues that are less favored in construction of URPs include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. URP sequences can be rich in glycine but URP sequences can also be rich in the amino acids glutamate, aspartate, serine, threonine, alanine or proline. Thus the predominant amino acid may be G, E, D, S, T, A or P. The inclusion of proline residues tends to reduce sensitivity to proteolytic degradation.
- The inclusion of hydrophilic residues typically increases URPs' solubility in water and aqueous media under physiological conditions. As a result of their amino acid composition, URP sequences have a low tendency to form aggregates in aqueous formulations and the fusion of URP sequences to other proteins or peptides tends to enhance their solubility and reduce their tendency to form aggregates, which is a separate mechanism to reduce immunogenicity.
- URP sequences can be designed to avoid certain amino acids that confer undesirable properties to the protein. For instance, one can design URP sequences to contain few or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation).
- Glycine-Rich URPs:
- In one embodiment, the subject URP comprises a glycine rich sequence (GRS). For example, glycine can be present predominantly such that it is the most prevalent residues present in the sequence of interest. In another example, URP sequences can be designed such that glycine residues constitute at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the total amino acids. URPs can also contain 100% glycines. In yet another example, the URPs contain at least 30% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 20%. In still another example, the URPs contain at least 40% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 10%. In still yet another example, the URPs contain at least about 50% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 5%.
- The length of GRS can vary between about 5 amino acids and 200 amino acids or more. For example, the length of a single, contiguous GRS can contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 240, 280, 320 or 400 or more amino acids. GRS may comprise glycine residues at both ends.
- GRS can also have a significant content of other amino acids, for example Ser, Thr, Ala, or Pro. GRS can contain a significant fraction of negatively charged amino acids including but not limited to Asp and Glu. GRS can contain a significant fraction of positively charged amino acids including but not limited to Arg or Lys. Where desired, URPs can be designed to contain only a single type of amino acid (i.e., Gly or Glu), sometimes only a few types of amino acid, e.g., two to five types of amino acids (e.g., selected from G, E, D, S, T, A and P), in contrast to typical proteins and typical linkers which generally are composed of most of the twenty types of amino acids. URPs may contain negatively charged residue's (Asp, Glu) in 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of the amino acids positions.
- Typically, the subject GRS-containing URP has about 30, 40, 50, 60, 70, 80, 90, 100, or more contiguous amino acids. When incorporated into a protein, the URP can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs. Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 30 amino acids. Preferably, the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, or more amino acids.
- The GRS-containing URPs are of particular interest due to, in part, the increased conformational freedom of glycine-containing peptides. Denatured peptides in solution have a high degree of conformational freedom. Most of that conformational freedom is lost upon binding of said peptides to a target like a receptor, an antibody, or a protease. This loss of entropy needs to be offset by the energy of interaction between the peptide and its target. The degree of conformational freedom of a denatured peptide is dependent on its amino acid sequences. Peptides containing many amino acids with small side chains tend to have more conformational freedom than peptides that are composed of amino acids with larger side chains. Peptides containing the amino acid glycine have particularly large degrees of freedom. It has been estimated that glycine-containing peptide bonds have about 3.4 times more entropy in solution as compared to corresponding alanine-containing sequences (D'Aquino, J. A., et al. (1996) Proteins, 25: 143-56). This factor increases with the number of glycine residues in a sequence. As a result, such peptides tend to lose more entropy upon binding to targets, which reduces their overall ability to interact with other proteins as well as their ability to adopt defined three-dimensional structures. The large conformational flexibility of glycine-peptide bonds is also evident when analyzing Ramachandran plots of protein structures where glycine peptide bonds occupy areas that are rarely occupied by other peptide bonds (Venkatachalam, C. M., et al. (1969) Annu Rev Biochem, 38: 45-82). Stites et al. studied a database of 12,320 residues from 61 nonhomologous, high resolution crystal structures to determine the phi, psi conformational preferences of each of the 20 amino acids. The observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributions were used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by −0.82+/−0.08 kcal/mol at 20° C. (Stites, W. E., et al. (1995) Proteins, 22: 132). These observations confirm the special role of glycine among the 20 natural amino acids.
- In designing the subject URPs, natural or non-natural sequences can be used. For example, a host of natural sequences containing high glycine content is provided in Table 1, Table 2, Table 3, and Table 4. One skilled in the art may adopt any one of the sequences as an URP, or modify the sequences to achieve the intended properties. Where immunogenicity to the host subject is of concern, it is preferable to design GRS-containing URPs based on glycine rich sequences derived from the host. Preferred GRS-containing URPs are sequences from human proteins or sequences that share substantial homology to the corresponding glycine rich sequences in the reference human proteins.
-
TABLE 1 Structural analysis of proteins that contain glycine rich sequences PDB sequences file Protein function Glycine rich 1K3V Porcine Parvovirus capsid sgggggggggrgagg 1FPV Feline Panleukopenia Virus tgsgngsgggggggsgg 1IJS CpV strain D, mutant A300d tgsgngsgggggggsgg 1MVM Mvm (strain I) virus ggsggggsgggg -
TABLE 2 Open reading frames encoding GRS with 300 or more glycine residues Gly GRS Gene Accession Organism (%) length length Predicted Function NP_974499 Arabidopsis thaliana 64 509 579 unknown ZP_00458077 Burkholderia cenocopacia 66 373 518 putative lipoprotein XP_477841 Oryza sativa 74 371 422 unknown NP_910409 Oryza sativa 75 368 400 putative cell-wall precursor NP_610660 Drosophila melanogaster 66 322 610 transposable element -
TABLE 3 Examples of human GRS Gly GRS Gene Hydro- Accession (%) length length phobics Predicted Function NP_000217 62 135 622 yes keratin 9 NP_631961 61 73 592 yes TBP-associated factor 15 isoform 1NP_476429 65 70 629 yes keratin 3 NP_000418 70 66 316 yes loricrin, cell envelope NP_056932 60 66 638 yes cytokeratin 2 -
TABLE 4 Additional examples of human GRS Number of Accession Sequences amino acids NP_006228. GPGGGGGPGGGGGPGGGGPGGGGGGGP 37 GGGGGGPGGG NP_787059 GAGGGGGGGGGGGGGSGGGGGGGGAGA 33 GGAGAG NP_009060 GGGSGSGGAGGGSGGGSGSGGGGGGAG 32 GGGGG NP_031393 GDGGGAGGGGGGGGSGGGGSGGGGGGG 27 NP_005850 GSGSGSGGGGGGGGGGGGSGGGGGG 25 NP_061856 GGGRGGRGGGRGGGGRGGGRGGG 22 NP_787059 GAGGGGGGGGGGGGGSGGGGGGGGAGA 33 GGAGAG NP_009060 GGGSGSGGAGGGSGGGSGSGGGGGGAG 32 GGGGG NP_031393 GDGGGAGGGGGGGGSGGGGSGGGGGGG 27 NP_115818 GSGGSGGSGGGPGPGPGGGGG 21 XP_376532 GEGGGGGGEGGGAGGGSG 18 NP_065104 GGGGGGGGDGGG 12 GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGGSSGGGSGTAGGHSG POU domain, class 4, transcription factor 1 [Homo sapiens] GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG YEATS domain containing 2 [Homo sapiens] GGSGAGGGGGGGGGGGSGSGGGGSTGGGGGTAGGG AT rich interactive domain 1B (SWI1-like) isoform 3; BRG1-binding protein ELD/OSA1; Eld (eyelid)/Osa protein [Homo sapiens] GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG AT rich interactive domain 1B (SWI1-like) isoform 2; BRG1-binding protein ELD/OSA1; Eld (eyelid)/Osa protein [Homo sapiens] GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG AT rich interactive domain 1B (SWI1-like) isoform 1; BRG1-binding protein ELD/OSA1; Eld (eyelid)/Osa protein [Homo sapiens] GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG purine-rich element binding protein A; purine-rich single-stranded DNA-binding protein alpha; transcriptional activator protein PUR-alpha [Homo sapiens] GHPGSGSGSGGGGGGGGGGGGSGGGGGGAPGG regulatory factor X1; trans-acting regulatory factor 1; enhancer factor C; MHC class II regulatory factor RFX [Homo sapiens] GGGGSGGGGGGGGGGGGGGSGSTGGGGSGAG bromo domain-containing protein disrupted in leukemia [Homo sapiens GGRGRGGRGRGSRGRGGGGTRGRGRGRGGRG unknown protein [Homo sapiens] GSGGSGGSGGGPGPGPGGGGGPSGSGSGPG PREDICTED: hypothetical protein XP_059256 [Homo sapiens] GGGGGGGGGGGRGGGGRGGGRGGGGEGGG zinc finger protein 281; ZNP-99 transcription factor [Homo sapiens] GGGGTGSSGGSGSGGGGSGGGGGGGSSG RNA binding protein (autoantigenic, hnRNP- associated with lethal yellow) short isoform; RNA- binding protein (autoantigenic); RNA-binding protein (autoantigenic, hnRNP-associated with lethal yellow) [Homo sapiens] GDGGGAGGGGGGGGSGGGGSGGGGGGG signal recognition particle 68kDa [Homo sapiens] GGGGGGGSGGGGGSGGGGSGGGRGAGG KIAA0265 protein [Homo sapiens] GGGAAGAGGGGSGAGGGSGGSGGRGTG engrailed homolog 2; Engrailed-2 [Homo sapiens GAGGGRGGGAGGEGGASGAEGGGGAGG RNA binding protein (autoantigenic, hnRNP- associated with lethal yellow) long isoform; RNA- binding protein (autoantigenic); RNA-binding protein (autoantigenic, hnRNP-associated with lethal yellow) [Homo sapiens] GDGGGAGGGGGGGGSGGGGSGGGGGGG androgen receptor; dihydrotestosterone receptor [Homo sapiens] GGGGGGGGGGGGGGGGGGGGGGGEAG homeo box D11; homeo box 4F; Hox-4.6, mouse, homolog of; homeobox protein Hox-D11 [Homo sapiens] GGGGGGSAGGGSSGGGPGGGGGGAGG frizzled 8; frizzled (Drosophila) homolog 8 [Homo sapiens] GGGGGPGGGGGGGPGGGGGPGGGGG ocular development-associated gene [Homo sapiens] GRGGAGSGGAGSGAAGGTGSSGGGG homeo box B3; homeo box 2G; homeobox protein Hox-B3 [Homo sapiens] GGGGGGGGGGGSGGSGGGGGGGGGG chromosome 2 open reading frame 29 [Homo sapiens] GGSGGGRGGASGPGSGSGGPGGPAG DKFZP564F0522 protein [Homo sapiens] GGHHGDRGGGRGGRGGRGGRGGRAG PREDICTED: similar to Homeobox even-skipped homolog protein 2 (EVX-2) [Homo sapiens GSRGGGGGGGGGGGGGGGGAGAGGG ras homolog gene family, member U; Ryu GTPase; Wnt-1 responsive Cdc42 homolog; 2310026M05Rik; GTP-binding protein like 1; CDC42-like GTPase [Homo sapiens] GGRGGRGPGEPGGRGRAGGAEGRG scratch 2 protein; transcriptional repressor scratch 2; scratch (drosophila homolog) 2, zinc finger protein [Homo sapiens] GGGGGDAGGSGDAGGAGGRAGRAG nucleolar protein family A, member 1; GAR1 protein [Homo sapiens] GGGRGGRGGGRGGGGRGGGRGGG keratin 1; Keratin-1; cytokeratin 1; hair alpha protein [Homo sapiens] GGSGGGGGGSSGGRGSGGGSSGG hypothetical protein FLJ31413 [Homo sapiens] GSGPGTGGGGSGSGGGGGGSGGG one cut domain, family member 2; onecut 2 [Homo sapiens] GARGGGSGGGGGGGGGGGGGGPG POU domain, class 3, transcription factor 2 [Homo sapiens] GGGGGGGGGGGGGGGGGGGGGDG PREDICTED: similar to THO complex subunit 4 (Tho4) (RNA and export factor binding protein 1) (REF 1-I) (Ally of AML-1 and LEF-1) (Aly/REF) [Homo sapiens] GGTRGGTRGGTRGGDRGRGRGAG PREDICTED: similar to THO complex subunit 4 (Tho4) (RNA and export factor binding protein 1) (REF 1-I) (Ally of AML-1 and LEF-1) (Aly/REF) [Homo sapiens] GGTRGGTRGGTRGGDRGRGRGAG POU domain, class 3, transcription factor 3 [Homo sapiens] GAGGGGGGGGGGGGGGAGGGGGG nucleolar protein family A, member 1; GAR1 protein [Homo sapiens] GGGRGGRGGGRGGGGRGGGRGGG fibrillarin; 34-kD nucleolar scleroderma antigen; RNA, U3 small nucleolar interacting protein 1 [Homo sapiens] GRGRGGGGGGGGGGGGGRGGGG zinc finger protein 579 [Homo sapiens] GRGRGRGRGRGRGRGRGRGGAG calpain, small subunit 1; calcium-activated neutral proteinase; calpain, small polypeptide; calpain 4, small subunit (30K); calcium-dependent protease, small subunit [Homo sapiens] GAGGGGGGGGGGGGGGGGGGGG keratin 9 [Homo sapiens] GGGSGGGHSGGSGGGHSGGSGG forkhead box D1; forkhead-related activator 4; Forkhead, drosophila, homolog-like 8; forkhead (Drosophila)-like 8 [Homo sapiens] GAGAGGGGGGGGAGGGGSAGSG PREDICTED: similar to RIKEN cDNA C230094B15 [Homo sapiens] GGPGTGSGGGGAGTGGGAGGPG GGGGGGGGGAGGAGGAGSAGGG cadherin 22 precursor; ortholog of rat PB-cadherin [Homo sapiens] GGDGGGSAGGGAGGGSGGGAG AT-binding transcription factor 1; AT motif- binding factor 1 [Homo sapiens] GGGGGGSGGGGGGGGGGGGGG eomesodermin; t box, brain, 2; eomesodermin (Xenopus laevis) homolog [Homo sapiens] GPGAGAGSGAGGSSGGGGGPG phosphatidylinositol transfer protein, membrane- associated 2; PYK2 N-terminal domain-interacting receptor 3; retinal degeneration B alpha 2 (Drosophila) [Homo sapiens] GGGGGGGGGGGSSGGGGSSGG sperm associated antigen 8 isoform 2; sperm membrane protein 1 [Homo sapiens] GSGSGPGPGSGPGSGPGHGSG PREDICTED: RNA binding motif protein 27 [Homo sapiens] GPGPGPGPGPGPGPGPGPGPG AP1 gamma subunit binding protein 1 isoform 1; gamma-synergin; adaptor-related protein complex 1 gamma subunit-binding protein 1 [Homo sapiens] GAGSGGGGAAGAGAGSAGGGG AP1 gamma subunit binding protein 1 isoform 2; gamma-synergin; adaptor-related protein complex 1 gamma subunit-binding protein 1 [Homo sapiens] GAGSGGGGAAGAGAGSAGGGG ankyrin repeat and sterile alpha motif domain containing 1; ankyrin repeat and SAM domain containing 1 [Homo sapiens] GGGGGGGSGGGGGGSGGGGGG methyl-CpG binding domain protein 2 isoform 1 [Homo sapiens] GRGRGRGRGRGRGRGRGRGRG triple functional domain (PTPRF interacting) [Homo sapiens] GGGGGGGSGGSGGGGGSGGGG forkhead box D3 [Homo sapiens GGEEGGASGGGPGAGSGSAGG sperm associated antigen 8 isoform 1; sperm membrane protein 1 [Homo sapiens] GSGSGPGPGSGPGSGPGHGSG methyl-CpG binding domain protein 2 testis- specific isoform [Homo sapiens] GRGRGRGRGRGRGRGRGRGRG cell death regulator aven; programmed cell death 12 [Homo sapiens] GGGGGGGGDGGGRRGRGRGRG regulator of nonsense transcripts 1; delta helicase; up-frameshift mutation 1 homolog (S. cerevisiae); nonsense mRNA reducing factor 1; yeast Upflp homolog [Homo sapiens] GGPGGPGGGGAGGPGGAGAG small conductance calcium-activated potassium channel protein 2 isoform a; apamin-sensitive small-conductance Ca2+-activated potassium channel [Homo sapiens] GTGGGGSTGGGGGGGGSGHG SRY (sex determining region Y)-box 1; SRY-related HMG-box gene 1 [Homo sapiens] GPAGAGGGGGGGGGGGGGGG transcription factor 20 isoform 2; stromelysin-1 platelet-derived growth factor-responsive element binding protein; stromelysin 1 PDGF-responsive element-binding protein; SPRE-binding protein; nuclear factor SPBP [Homo sapiens] GGTGGSSGSSGSGSGGGRRG transcription factor 20 isoform 1; stromelysin-1 platelet-derived growth factor-responsive element binding protein; stromelysin 1 PDGF-responsive element-binding protein; SPRE-binding protein; nuclear factor SPBP [Homo sapiens] GGTGGSSGSSGSGSGGGRRG Ras-interacting protein 1 [Homo sapiens] GSGTGTTGSSGAGGPGTPGG BMP-2 inducible kinase isoform b [Homo sapiens] GGSGGGAAGGGAGGAGAGAG BMP-2 inducible kinase isoform a [Homo sapiens] GGSGGGAAGGGAGGAGAGAG forkhead box C1; forkhead-related activator 3; Forkhead, drosophila, homolog-like 7; forkhead (Drosophila)-like 7; iridogoniodysgenesis type 1 [Homo sapiens] GSSGGGGGGAGAAGGAGGAG splicing factor p54; arginine-rich 54 kDa nuclear protein [Homo sapiens] GPGPSGGPGGGGGGGGGGGG v-maf musculoaponeurotic fibrosarcoma oncogene homolog; Avian musculoaponeurotic fibrosarcoma (MAF) protooncogene; v-maf musculoaponeurotic fibrosarcoma (avian) oncogene homolog [Homo sapiens] GGGGGGGGGGGGGGAAGAGG small nuclear ribonucleoprotein D1 polypeptide 16kDa; snRNP core protein D1; Sm-D autoantigen; small nuclear ribonucleoprotein D1 polypeptide (16kD) [Homo sapiens] GRGRGRGRGRGRGRGRGRGG hypothetical protein H41 [Homo sapiens] GSAGGSSGAAGAAGGGAGAG - The sequences of non-glycine residues in these GRS can be selected to optimize the properties of URPs and hence the proteins that contain the desired URPs. For instance, one can optimize the sequences of URPs to enhance the selectivity of the resulting protein for a particular tissue, specific cell type or cell lineage. For example, one can incorporate protein sequences that are not ubiquitously expressed, but rather are differentially expressed in one or more of the body tissues including heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, and selected tissues that are affected by diseases such as infectious diseases, autoimmune disease, renal, neronal, cardiac disorders and cancers. One can employ sequences representative of a specific developmental origin, such as those expressed in an embryo or an adult, during ectoderm, endoderm or mesoderm formation in a multi-cellular organism. One can also utilize sequence involved in a specific biological process, including but not limited to cell cycle regulation, cell differentiation, apoptosis, chemotaxsis, cell motility and cytoskeletal rearrangement. One can also utilize other non-ubiquitously expressed protein sequences to direct the resulting protein to a specific subcellular locations: extracellular matrix, nucleus, cytoplasm, cytoskeleton, plasma and/or intracellular membranous structures which include but are not limited to coated pits, Golgi apparatus, endoplasmic reticulum, endosome, lysosome, and mitochondria.
- A variety of these tissue-specific, cell-type specific, subcellular location specific sequences are known and available from numerous protein databases. Such selective URP sequences can be obtained by generating libraries of random or semi-random URP sequences, injecting them into animals or patients, and determining sequences with the desired tissue selectivity in tissue samples. Sequence determination can be performed by mass spectrometry. Using similar methods one can select URP sequences that facilitate oral, buccal, intestinal, nasal, thecal, peritoneal, pulmonary, rectal, or dermal uptake.
- Of particular interest are URP sequences that contain regions that are relatively rich in the positively charged amino acids arginine or lysine which favor cellular uptake or transport through membranes. URP sequences can be designed to contain one or several protease-sensitive sequences. Such URP sequences can be cleaved once the product of the invention has reached its target location. This cleavage may trigger an increase in potency of the pharmaceutically active domain (pro-drug activation) or it may enhance binding of the cleavage product to a receptor. URP sequences can be designed to carry excess negative charges by introducing aspartic acid or glutamic acid residues. Of particular interest are URP that contain great than 5%, greater than 6%, 7%, 8%, 9%, 10%, 15%, 30% or more glutamic acid and less than 2% lysine or arginine. Such URPs carry an excess negative charge and as a result they have a tendency to adopt open conformations due to electrostatic repulsion between individual negative charges of the peptide. Such an excess negative charge leads to an effective increase in their hydrodynamic radius and as a result it can lead to reduced kidney clearance of such molecules. Thus, one can modulate the effective net charge and hydrodynamic radius of a URP sequence by controlling the frequency and distribution of negatively charged amino acids in the URP sequences. Most tissues and surfaces in a human or animal carry excess negative charges. By designing URP sequences to carry excess negative charges one can minimize non-specific interactions between the resulting protein comprising the URP and various surfaces such as blood vessels, healthy tissues, or various receptors.
- URPs may have a repetitive amino acid sequence of the format (Motif)x in which a sequence motif forms a direct repeat (ie ABCABCABCABC) or an inverted repeat (ABCCBAABCCBA) and the number of these repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 50 or more. URPs or the repeats inside URPs often contain only 1, 2, 3, 4, 5 or 6 different types of amino acids. URPs typically consist of repeats of human amino acid sequences that are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36 or more amino acids long, but URPs may also consist of non-human amino acid sequences that are 20, 22, 24, 26, 28, 30, 32, 34 36, 38 40, 42, 44, 46, 48, 50 amino acids long.
- URPs Derived from Human Sequences:
- URPs can be derived from human sequences. The human genome contains many subsequences that are rich in one particular amino acid. Of particular interest are such amino acid sequences that are rich in a hydrophilic amino acid like serine, threonine, glutamate, aspartate, or glycine. Of particular interest are such subsequences that contain few hydrophobic amino acids. Such subsequences are predicted to be unstructured and highly soluable in aqeuous solution. Such human subsequences can be modified to further improve their utility.
FIG. 17 shows an exemplary human sequence that is rich in serine and that can be isolated as the subject URP. The exemplified dentin sialophosphoprotein contains a 670-amino acid subsequence in which 64% of the residues are serine and most other positions are hydrophilic amino acids such as aspartate, asparagines, and glutamate. The sequence is extremely repetitive and as a result it has a low information content. One can directly use subsequences of such a human protein. Where desired, one can modify the sequence in a way that preserves its overall character but which makes it more suitable for pharmaceutical applications. Examples of sequences that are related to dentin sialophosphoprotein are (SSD)n, (SSDSSN)n, (SSE)n, where n is between about 4 and 200. - The use of sequences from human proteins is particularly desirable in design of URPs with reduced immunogenicity in a human subject. A key step for eliciting an immune response to a foreign protein is the presentation of peptide fragments of said protein by MHC class II receptors. These MHCII-bound fragments can then be detected by T cell receptors, which triggers the proliferation of T helper cells and initiates an immune response. The elimination of T cell epitopes from pharmaceutical proteins has been recognized as a means to reduce the risk of eliciting an immune reaction (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). MHCII receptors typically interact with an epitope having e.g., a 9-amino acid long region of the displayed peptides. Thus, one can reduce the risk of eliciting an immune response to a protein in patients if all or most of the possible 9mer subsequences of the protein can be found in human proteins and if so, these sequences and repeats of these sequences will not be recognized by the patient as foreign sequences. One can incorporate human sequences into the design of URP sequences by oligomerizing or concatenating human sequences that have suitable amino acid compositions. These can be direct repeats or inverted repeats or mixtures of different repeats. For instance one can oligomerize the sequences shown in table 2. Such oligomers have reduced risk of being immunogenic. However, the junction sequences between the monomer units can still contain T cell epitopes that can trigger an immune reaction, which is illustrated in
FIG. 3 . One can further reduce the risk of eliciting an immune response by designing URP sequences based on multiple overlapping human sequences. This approach is illustrated inFIG. 4 . The URP sequence inFIG. 2 designed as an oligomer based on multiple human sequences such that each 9mer subsequences of the oligomer can be found in a human protein. In these designs, every 9-mer subsequence is a human sequence. An example of a URP sequence based on three human sequences is shown inFIG. 5 . It is also possible to design URP sequences based on a single human sequences such that all possible 9mer subsequences in the oligomeric URP sequences occur in the same human protein. An example is shown inFIG. 6 based on the POU domain that is rich in glycine and proline. The repeating monomer in the URP sequence is only a fragment of the human protein and its flanking sequences is identical to the repeating unit as illustrated inFIG. 6 . Non-oligomeric URP sequences can be designed based on human proteins as well. The primary conditions are that all 9mer sub-sequences can be found in human sequences. The amino acid composition of the sequences preferably contains few hydrophobic residues. Of particular interest are URP sequences that are designed based on human sequences and that contain a large fraction of glycine residues. - Utilizing this or similar scheme, one can design a class of URPs that comprise repeat sequences with low immunogenicity to the host of interest. Host of interest can be any animals, including vertebrates and invertebrates. Preferred hosts are mammals such as primates (e.g. chimpanzees and humans), cetaceans (e.g. whales and dolphins), chiropterans (e.g. bats), perrisodactyls (e.g. horses and rhinoceroses), rodents (e.g. rats), and certain kinds of insectivores such as shrews, moles and hedgehogs. Where human is selected as the host, the URPs typically contain multiple copies of the repeat sequences or units, wherein the majority of segments comprising about 6 to about 15 contiguous amino acids are present in one or more native human proteins. One can also design URPs in which the majority of segments comprising between about 9 to about 15 contiguous amino acids are found in one or more native human proteins. As used herein, majority of the segments refers to more than about 50%, preferably 60%, preferably 70%, preferably 80%, preferably 90%, preferably 100%. Where desired, each of the possible segments between about 6 to 15 amino acids, preferably between about 9 to 15 amino acids within the repeating units are present in one or more native human proteins. The URPs can comprise multiple repeating units or sequences, for example having 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeating units.
- Design of URPs that are Substantially Free of Human T-Cell Epitopes:
- URP sequences can be designed to be substantially free of epitopes recognized by human T cells. For instance, one can synthesize a series of semi-random sequences with amino acid compositions that favor denatured, unstructured conformations and evaluate these sequences for the presence of human T cell epitopes and whether they are human sequences. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and in particular 9-mer subsequences that are not human. An alternative is to evaluate multiple peptide sequences that can be assembled into repeating units as described in the previous section for the assembly of human sequences. Another alternative is to design URP sequences that result in low scores using epitope prediction algorithms like TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61). Another approach to avoiding T-cell epitopes is to avoid amino acids that can serve as anchor residues during peptide display on MHC, such as M, I, L, V, F. Hydrophobic amino acids and positively charged amino acids can frequently serve as such anchor residues and minimizing their frequency in a URP sequences reduces the chance of generating T-cell epitopes and thus eliciting an immune reaction. The selected URPs generally contain subsequences that are found in at least one human protein, and have a lower content of hydrophobic amino acids.
- URP sequences can be designed to optimize protein production. This can be achieved by avoiding or minimizing repetitiveness of the encoding DNA. URP sequences such as poly-glycine may have very desirable pharmaceutical properties but their manufacturing can be difficult due to the high GC-content of DNA sequences encoding for GRS and due to the presence of repeating DNA sequences that can lead to recombination.
- As noted above, URP sequences can be designed to be highly repetitive at the amino acid level. As a result the URP sequences have very low information content and the risk of eliciting an immune reaction can be reduced.
- Non-limiting examples of URPs containing repeating amino acids are: poly-glycine, poly-glutamic acid, poly-aspartic acid, poly-serine, poly-threonine, (GX)n where G is glycine and X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 20, (GGX)n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 13, (GGGX)n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 10, (GGGGX)n where X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 8, (G2X)n where X is serine, aspartic acid, glutamic acid, threonine, or proline, n is at least 15, and z is between 1 and 20.
- The number of these repeats can be any number between 10 and 100. Products of the invention may contain URP sequences that are semi-random sequences. Examples are semi-random sequences containing at least 30, 40, 50, 60 or 70% glycine in which the glycines are well dispersed and in which the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 70, 60, 50, 40, 30, 20, or 10% when combined. A preferred semi-random URP sequence contains at least 40% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 10%. A more preferred random URP sequence contains at least 50% glycine and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine, and isoleucine is less then 5%. URP sequences can be designed by combining the sequences of two or more shorter URP sequences or fragments of URP sequences. Such a combination allows one to better modulate the pharmaceutical properties of the product containing the URP sequences and it allows one to reduce the repetitiveness of the DNA sequences encoding the URP sequences, which can improve expression and reduce recombination of the URP encoding sequences.
- URP sequences can be designed and selected to possess several of the following desired properties: a) high genetic stability of the coding sequences in the production host, b) high level of expression, c) low (predicted/calculated) immunogenicity, d) high stability in presence of serum proteases and/or other tissue proteases, e) large hydrodynamic radius under physiological conditions. One exemplary approach to obtain URP sequences that meet multiple criteria is to construct a library of candidate sequences and to identify from the library the suitable subsequences. Libraries can comprise random and/or semi-random sequences. Of particular utility are codon libraries, which is a library of DNA molecules that contains multiple codons for the identical amino acid residue. Codon randomization can be applied to selected amino acid positions of a certain type or to most or all positions. True codon libraries encode only a single amino acid sequence, but they can easily be combined with amino acid libraries, which is a population of DNA molecules encoding a mixture of (related or unrelated) amino acids at the same residue position. Codon libraries allow the identification of genes that have relatively low repetitiveness at the DNA level but that encode highly repetitive amino acid sequences. This is useful because repetitive DNA sequences tend to recombine, leading to instability. One can also construct codon libraries that encode limited amino acid diversity. Such libraries allow introduction of a limited number of amino acids in some positions of the sequence while other positions allow for codon variation but all codons encode the same amino acid. One can synthesize partially random oligonucleotides by incorporating mixtures of nucleotides at the same position during oligonucleotide synthesis. Such partially random oligonucleotides can be fused by overlap PCR or ligation-based approaches. In particular, one can multimerize semi-random oligonucleotides that encode glycine-rich sequences. These oligonucleotides can differ in length and sequences and codon usage. As a result, one obtains a library of candidate URP sequences. Another method to generate libraries is to synthesize a starting sequence and subsequently subject said sequence to partial randomization. This can be done by cultivation of the gene encoding the URP sequences in a mutator strain or by amplification of the encoding gene under mutagenic conditions (Leung, D., et al. (1989) Technique, 1: 11-15). URP sequences with desirable properties can be identified from libraries using a variety of methods. Sequences that have a high degree of genetic stability can be enriched by cultivating the library in a production host. Sequences that are unstable will accumulate mutations, which can be identified by DNA sequencing. Variants of URP sequences that can be expressed at high level can be identified by screening or selection using multiple protocols known to someone skilled in the art. For instance one can cultivate multiple isolates from a library and compare expression levels. Expression levels can be measured by gel analysis, analytical chromatography, or various ELISA-based methods. The determination of expression levels of individual sequence variants can be facilitated by fusing the library of candidate URP sequences to sequence tags like myc-tag, His-tag, HA-tag. Another approach is to fuse the library to an enzyme or other reporter protein like green fluorescent protein. Of particular interest is the fusion of the library to a selectable marker like beta-lactamase or kanamycin-acyl transferase. One can use antibiotic selection to enrich for variants with high level of expression and good genetic stability. Variants with good protease resistance can be identified by screening for intact sequences after incubation with proteases. An effective way to identify protease-resistant URP sequences is bacterial phage display or related display methods. Multiple systems have been described where sequences that undergo rapid proteolysis can be enriched by phage display. These methods can be easily adopted to enrich for protease resistant sequences. For example, one can clone a library of candidate URP sequences between an affinity tag and the pIII protein of M13 phage. The library can then be exposed to proteases or protease-containing biological samples like blood or lysosomal preparations. Phage that contain protease-resistant sequences can be captured after protease treatment by binding to the affinity tag. Sequences that resist degradation by lysosomal preparations are of particular interest because lysosomal degradation is a key step during antigen presentation in dendritic and other antigen presenting cells. Phage display can be utilized to identify candidate URP sequences that do not bind to a particular immune serum in order to identify URP sequences with low immunogenicity. One can immunize animals with a candidate URP sequence or with a library of URP sequences to raise antibodies against the URP sequences in the library. The resulting serum can then be used for phage panning to remove or identify sequences that are recognized by antibodies in the resulting immune serum. Other methods like bacterial display, yeast display, ribosomal display can be utilized to identify variants of URP sequences with desirable properties. Another approach is the identification of URP sequences of interest by mass spectrometry. For instance, one can incubate a library of candidate URP sequences with a protease or biological sample of interest and identify sequences that resist degradation by mass spectrometry. In a similar approach one can identify URP sequences that facilitate oral uptake. One can feed a mixture of candidate URP sequences to animals or humans and identify variants with the highest transfer or uptake efficiency across some tissue barrier (ie dermal, etc) by mass spectrometry. In a similar way, one can identify URP sequences that favor other uptake mechanisms like pulmonary, intranasal, rectal, transdermal delivery. One can also identify URP sequences that favor cellular uptake or URP sequences that resist cellular uptake.
- URP sequences can be designed by combining URP sequences or fragments of URP sequences that were designed by any of the methods described above. In addition, one can apply semi-random approaches to optimize sequences that were designed based on the rules described above. Of particular interest is codon optimization with the goal of improving expression of the enhanced proteins and to improve the genetic stability of the encoding gene in the production hosts. Codon optimization is of particular importance for URP sequences that are rich in glycine or that have very repetitive amino acid sequences. Codon optimization can be performed using computer programs (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53), some of which minimize ribosomal pausing (Coda Genomics Inc.). When designing URP sequences one can consider a number of properties. One can minimize the repetitiveness in the encoding DNA sequences. In addition, one can avoid or minimize the use of codons that are rarely used by the production host (ie the AGG and AGA arginine codons and one Leucine codon in E. coli) DNA sequences that have a high level of glycine tend to have a high GC content that can lead to instability or low expression levels. Thus, when possible it is preferred to choose codons such that the GC-content of URP-encoding sequence is suitable for the production organism that will be used to manufacture the URP.
- URP encoding genes can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension. URP modules can be constructed such that the URP module-encoding gene has low repetitiveness while the encoded amino acid sequence has a high degree of repetitiveness. The approach is illustrated in
FIG. 11 . As a first step, one constructs a library of relatively short URP sequences. This can be a pure codon library such that each library member has the same amino acid sequence but many different coding sequences are possible. To facilitate the identification of well-expressing library members one can construct the library as fusion to a reporter protein. Examples of suitable reporter genes are green fluorescent protein, luciferace, alkaline phosphatase; beta-galactosidase. By screening one can identify short URP sequences that can be expressed in high concentration in the host organism of choice. Subsequently, one can generate a library of random URP dimers and repeat the screen for high level of expression. Dimerization can be performed by ligation, overlap extension or similar cloning techniques. This process of dimerization and subsequent screening can be repeated multiple times until the resulting URP sequence has reached the desired length. Optionally, one can sequence clones in the library to eliminate isolates that contain undesirable sequences. The initial library of short URP sequences can allow some variation in amino acid sequence. For instance one can randomize some codons such that a number of hydrophilic amino acids can occur in said position. During the process of iterative multimerization one can screen library members for other characteristics like solubility or protease resistance in addition to a screen for high-level expression. Instead of dimerizing URP sequences one can also generate longer multimers. This allows one to faster increase the length of URP modules. - Many URP sequences contain particular amino acids at high fraction. Such sequences can be difficult to produce by recombinant techniques as their coding genes can contain repetitive sequences that are subject to recombination. Furthermore, genes that contain particular codons at very high frequencies can limit expression as the respective loaded tRNAs in the production host become limiting. An example is the recombinant production of GRS. Glycine residues are encoded by 4 triplets, GGG, GGC, GGA, and GGT. As a result, genes encoding GRS tend to have high GC-content and tend to be particularly repetitive. An additional challenge can result from codon bias of the production host. In the case of E. coli, two glycine codons, GGA and GGG, are rarely used in highly expressed proteins. Thus codon optimization of the gene encoding URP sequences can be very desirable. One can optimize codon usage by employing computer programs that consider codon bias of the production host (Gustafsson, C., et al. (2004) Trends Biotechnol, 22: 346-53). As an alternative, one can construct codon libraries where all members of the library encode the same amino acid sequence but where codon usage is varied. Such libraries can be screened for highly expressing and genetically stable members which are particularly suitable for the large-scale production of URP-containing products.
- As noted above, the subject URPs are particularly useful as modules for design of proteins of therapeutic value. Accordingly, the present invention provides proteins comprising one or more subject URPs. Such proteins are termed herein Multivalent Unstructured Recombinant Proteins (MURPs).
- To construct MURPs, one or more URP sequences can be fused to the N-terminus or C-terminus of a protein or inserted in the middle of the protein, e.g., into loops of a protein or in between modules of the protein of interest, to give the resulting modified protein improved properties relative to the unmodified protein. The combined length of URP sequences that are attached to a protein can be 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- The subject MURPs exhibit one or more improved properties as detailed below.
- Improved Half-Life:
- Adding a URP sequences to a pharmaceutically active protein can improve many properties of that protein. In particular, adding a long URP sequence can significantly increase the serum half-life of the protein. Such URPs typically contain amino acid sequences of at least about 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids.
- The URPs can be fragmented such that the resulting protein contains multiple URPs, or multiple fragments of URPs. Some or all of these individual URP sequences may be shorter that 40 amino acids as long as the combined length of all URP sequences in the resulting protein is at least 30 amino acids. Preferably, the resulting protein has a combined length of URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids. In one aspect, the fused URPS can increase the hydrodynamic radius of a protein and thus reduces its clearance from the blood by the kidney. The increase in the hydrodynamic radius of the resulting fusion protein relative to the unmodified protein can be detected by ultracentrifugation, size exclusion chromatography, or light scattering.
- Improved Tissue Selectivity:
- Increasing the hydrodynamic radius can also lead to reduced penetration into tissues, which can be exploited to minimize side effects of a pharmaceutically active protein. It is well documented that hydrophilic polymers have a tendency to accumulate selectively in tumor tissue which is caused by the enhanced permeability and retention (EPR) effect. The underlying cause of the EPR effect is the leaky nature of tumor vasculature (McDonald, D. M., et al. (2002) Cancer Res, 62: 5381-5) and the lack of lymphatic drainage in tumor tissues. Therefore, the selectivity of pharmaceutically active proteins for tumor tissues can be enhanced by adding hydrophilic polymers. As such, the therapeutic index of a given pharmaceutically active protein can be increased via incorporating the subject URPS.
- Protection from Degradation and Reduced Immunogenicity:
- Adding URP sequences can significantly improve the protease resistance of a protein. URP sequences themselves can be designed to be protease resistant and by attaching them to a protein one can shield that protein from the access of degrading enzymes. URP sequences can be added to pharmaceutically active proteins with the goal of reducing undesirable interactions of the protein with other receptors or surfaces. To achieve this, it can be beneficial to add the URP sequences to the pharmaceutically active protein in proximity to the site of the protein that makes such undesirable contacts. In particular, one can add URP sequences to pharmaceutically active proteins with the goal of reducing their interactions with any component of the immune system to prevent an immune response against the product of the invention. Adding a URP sequence to a pharmaceutically active protein can reduce interaction with pre-existing antibodies or B-cell receptors. Furthermore, the addition of URP sequences can reduce the uptake and processing of the product of the invention by antigen presenting cells. Adding one or more URP sequence to a protein is a preferred way of reducing its immunogenicity as it will suppress an immune response in many species allowing one to predict the expected immunogenicity of a product in patients based on animal data. Such species independent testing of immunogenicity is not possible for approaches that are based on the identification and removal of human T cell epitopes or sequences comparison with human sequences.
- Interruption of T Cell Epitopes:
- URP sequences can be introduced into proteins in order to interrupt T cell epitopes. This is particularly useful for proteins that combine multiple separate functional modules. The formation of T cell epitopes requires that peptide fragments of a protein antigen bind to MHC. MHC molecules interact with a short segment of amino acids typically 9 contiguous residues of the presented peptides. The direct fusion of different binding modules in a protein molecule can lead to T cell epitopes that span two neighboring domains. By separating the functional modules by URP modules prevents the generation of such module-spanning T cell epitopes as illustrated in
FIG. 7 . The insertion of URP sequences between functional modules can also interfere with proteolytic processing in antigen presenting cells, which will lead to an additional reduction of immunogenicity. Another approach to reduce the risk of immunogenicity is to disrupt T cell epitopes within functional modules of a product. In the case of microproteins, one approach is to have some of the intercysteine loops (those that are not involved in target binding) be glycine-rich. In microproteins, whose structure is due to a small number of cysteines, one could in fact replace most or all of the residues that are not involved in target binding with glycine, serine, glutamate, threonine, thus reducing the potential for immunogenicity while not affecting the affinity for the target. For instance, this can be carried out by performing a ‘glycine-scan’ of all residues, in which each residue is replaced by a glycine, then selecting the clones which retain target binding using phage display or screening, and then combining all of the glycine substitutions that are permitted. In general, functional modules have a much higher probability to contain T cell epitopes than URP modules. One can reduce the frequency of T cell epitopes in functional modules by replacing all or many non-critical amino acid residues with small hydrophilic residues like gly, ser, ala, glu, asp, asn, gln, thr. Positions in a functional module that allow replacement can be identified using a variety of random or structure based protein engineering approaches. - Improved Solubility:
- Functional modules of a protein can have limited solubility. In particular, binding modules tend to carry hydrophobic residues on their surface, which can limit their solubility and can lead to aggregation. By spacing or flanking such functional modules with URP modules one can improve the overall solubility of the resulting product. This is in particular true for URP modules that carry a significant percentage of hydrophilic or charged residues. By separating functional modules with soluble URP modules one can reduce intramolecular interactions between these functional modules
- Improved pH Profile and Homogeneity of Product Charge:
- URP sequences can be designed to carry an excess of negative or positive charges. As a result they confer an electrostatic field to any fusion partner which can be utilized to shift the pH profile of an enzyme or a binding interaction. Furthermore, the electrostatic field of a charged URP sequence can increase the homogeneity of pKa values of surface charges of a protein product, which leads to sharpened pH profiles of ligand interactions and to sharpened separations by isoelectric focusing or chromatofocusing.
- Improved Purification Properties Due to Sharper Product pKa:
- Each amino acid in solution by itself has a single, fixed pKa, which is the pH at which its functional groups are half protonated. In a typical protein you have many types of residues and due to proximity and protein breathing effects, they also change each other's effective pKa in variable ways. Because of this, at a wide range of pH conditions, typical proteins can adopt hundreds of differently ionized species, each with a different molecular weight and net charge, due to large numbers of combinations of charged and neutral amino acid residues. This is referred to as a broad ionization spectrum and makes the analysis (ie Mass Spec) and purification of such proteins more difficult.
- PEG is uncharged and does not affect the ionization spectrum of the protein it is attached to, leaving it with a broad ionization spectrum. However, a URP with a high content of Gly and Glu in principle exist in only two states: neutral (—COOH) when the pH is below the pKa of Glutamate and negatively charged (—COO−) when the pH is above the pKa of Glutamate. URP modules can form a single, homogeneously ionizated type of molecule and can yield a single mass in mass spectrometry.
- Where desired, MURPs can be expressed as a fusion with an URP having a single type of charge (Glu) distributed at constant spacing through the URP module. One may choose to incorporate 25-50 Glu residues per 20 kD of URP and all of these 25-50 residues would have very similar pKa.
- In addition, adding 25-50 negative charges to a small protein like IFN, hGH or GCSF (with only 20 charged residues) will increase the charge homogeneity of the product and sharpen its isoelectric point, which will be very close to the pKa of free glutamate.
- The increase in the homogeneity of the charge of the protein population has favorable processing properties, such as in ion exchange, isoelectric focusing, mass spec, etc. compared to traditional PEGylation.
- Improved Formulation and/or Delivery:
- Addition of URP sequences to pharmaceutically active proteins can significantly simplify the formulation and or the delivery of the resulting products. URP sequences can be designed to be very hydrophilic and as a result they improve the solubility of (for example) human proteins, which often contain hydrophobic patches that they use to bind to other human proteins. The formulation of such human proteins, like antibodies, can be quite challenging and often limits their concentration and delivery options. URPs can reduce product precipitation and aggregation and it allows one to use simpler formulations containing fewer ingredients, that are typically needed to stabilize a product in solution. The improved solubility of URP sequences-containing products allows to formulate these products at higher concentration and as a result one can reduce the injection volume for injectable products, which may enable home injection, which is limited to a very low injected volume. Addition of a URP sequence can also simplify the storage of the resulting formulated products. URP sequences can be added to pharmaceutically active proteins to facilitate their oral, pulmonary, rectal, or intranasal uptake. URP sequences can facilitate various modes of delivery because they allow higher product concentrations and improved product stability. Additional improvements can be achieved by designing URP sequences that facilitate membrane penetration.
- Improved Production:
- Adding URP sequences can have significant benefits for the production of the resulting product. Many recombinant products, especially native human proteins, have a tendency to form aggregates during production that can be difficult or impossible to dissolve and even when removed from the final product they may re-occur. These are usually due to hydrophobic patches by which these (native human) proteins contacted other (native human) proteins and mutating these residues is considered risky because of immunogenicity. However, URPs can increase the hydrophilicity of such proteins and enable their formulation without mutating the sequence of the human protein. URP sequences can facilitate the folding of a protein to reach its native state. Many pharmaceutically active proteins are produced by recombinant methods in a non-native aggregated state. These products need to be denatured and subsequently they are incubated under conditions that allow the proteins to fold into their native active state. A frequent side reaction during renaturation is the formation of aggregates. The fusion of URP sequences to a protein significantly reduces its tendency to form aggregates and thus it facilitates the folding of the pharmaceutically active component of the product. URP-containing products are much easier to prepare as compared to polymer-modified proteins. Chemical polymer-modification requires extra modification and purification steps after the active protein has been purified. In contrast, URP sequences can be manufactured using recombinant DNA methods together with the pharmaceutically active protein. The products of the invention are also significantly easier to characterize compared to polymer-modified products. Due to the recombinant production process one can obtain more homogeneous products with defined molecular characteristics. URP sequences can also facilitate the purification of a product. For instance URP sequences can include subsequences that can be captured by affinity chromatography. An example are sequences rich in histidine, which can be captured on resins with immobilized metals like nickel. URP sequences can also be designed to have an excess of negatively or positively charged amino acids. As a result they can significantly impact the net charge of a product, which can facilitate product purification by ion-exchange chromatography or preparative electrophoresis.
- The subject MURPs can contain a variety of modules, including but not limited to binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.
FIG. 1 depicts an exemplary MURP having multiple modules. However, MURPs can also have relatively simple architectures that are illustrated inFIG. 2 . MURPs can also contain fragmentation sites. These can be protease-sensitive sequences or chemically sensitive sequences that can be preferentially cleaved when the MURPs reach their target site. - Binding Module (BM):
- The MURPs of the present invention may comprise one or more binding modules. Binding module (BM) refers to a peptide or protein sequence that can bind specifically to one or several targets, which may be one or more therapeutic targets or accessory targets, such as for cell-, tissue- or organ targeting. BMs can be linear or cyclic peptides, cysteine-constrained peptides, microproteins, scaffold proteins (e.g., fibronectin, ankyrins, crystalline, streptavidin, antibody fragments, domain antibodies), peptidic hormones, growth factors, cytokines, or any type of protein domain, human or non-human, natural or non-natural, and they may be based on a natural scaffold or not based on a natural scaffold, or based on combinations or they may be fragments of any of the above. Optionally, these BMs can be engineered by adding, removing or replacing one or multiple amino acids in order to enhance their binding properties, their stability, or other properties. Binding modules can be obtained from natural proteins, by design or by genetic package display, including phage display, cellular display, ribosomal display or other display methods. Binding modules may bind to the same copy of the same target, which results in avidity, or they may bind to different copies of the same target (which can result in avidity if these copies are somehow connected or linked, such as by a cell membrane), or they may bind to two unrelated targets (which yields avidity if these targets are somehow linked, such as by a membrane). Binding modules can be identified by screening or otherwise analyzing random libraries of peptides or proteins.
- Particularly desirable binding modules are those that upon incorporation into a MURP, the MURP yield a desirable Tepitope score. The Tepitope score of a protein is the log of the Kd (dissociation constant, affinity, off-rate) of the binding of that protein to multiple of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 15 logs, from about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0, -1, -2, -3, -4, -5 (10e10 Kd) to about −5. Preferred MURPs yield a score less than about −3.5 [KKW: On absolute scale?]
- Of particular interest are also binding modules comprising disulfide bonds formed by pairing two cysteine residues. In certain embodiments, the binding modules comprise polypeptides having high cysteine content or high disulfide density (HDD). Binding modules of the HDD family typically have 5-50% (5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50%) cysteine residues and each domain typically contains at least two disulfides and optionally a co-factor such as calcium or another ion.
- The presence of HDD scaffold allows these modules to be small but still adopt a relatively rigid structure. Rigidity is important to obtain high binding affinities, resistance to proteases and heat, including the proteases involved in antigen processing, and thus contributes to the low or non-immunogenicity of these modules. The disulfide framework folds the modules without the need for a large number of hydrophobic side chain interactions in the interior of most modules. The small size is also advantageous for fast tissue penetration and for alternative delivery such as oral, nasal, intestinal, pulmonary, blood-brain-barrier, etc. In addition, the small size also helps to reduce immunogenicity. A higher disulfide density is obtainable, either by increasing the number of disulfides or by using domains with the same number of disulfides but fewer amino acids. It is also desirable to decrease the number of non-cysteine fixed residues, so that a higher percentage of amino acids is available for target binding.
- The cysteine-containing binding modules can adopt a wide range of disulfide bonding patterns (DBPs). For example, two-disulfide modules can have three different disulfide bonding patterns (DBPs), three-disulfide modules can have 15 different DBPs and four-disulfide modules have up to 105 different DBPs. Natural examples exist for all of the 2SS DBPs, the majority of the 3SS DBPs and less than half of the 4SS DBPs. In one aspect, the total number of disulfide bonding patterns can be calculated according to the formula:
-
- wherein n=the predicted number of disulfide bonds formed by the cysteine residues, and wherein Π represents the product of (2i-1), where i is a positive integer ranging from 1 up to n.
- Accordingly, in one embodiment, the modules used in MURPs are natural or non-naturally occurring cysteine (C)-containing scaffold exhibiting a binding specificity towards a target molecule, wherein the non-naturally occurring cysteine (C)-containing scaffold comprise intra-scaffold cysteines according to a pattern selected from the group of permutations represented by the formula
-
- wherein n equals to the predicted number of disulfide bonds formed by the cysteine residues, and wherein Π represents the product of (2i-1), where i is a positive integer ranging from 1 up to n. In one aspect, the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having two disulfide bonds formed by pairing cysteines contained in the polypeptide according to a pattern selected from the group consisting of C1-2, 3-4, C1-3, 2-4, and C1-4, 2-3, wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond. In another aspect, the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having three disulfide bonds formed by pairing intra-scaffold cysteines according to a pattern selected from the group consisting of C1-2, 3-4, 5-6, C1-2, 3-5, 4-6, C1-2, 3-6, 4-5, C1-3, 2-4, 5-6, C1-3, 2-5, 4-6, C1-3, 2-6, 4-5, C1-4, 2-3, 5-6, C1-4, 2-6, 3-5, C1-5, 2-3, 4-6, C1-5, 2-4, 3-6, C1-5, 2-6, 3-4, C1-6, 2-3, 4-5, and C1-6, 2-5, 3-4, wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond. In yet another aspect, the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having at least four disulfide bonds formed by pairing cysteines contained in the polypeptide according to a pattern selected from the group of permutations defined by the formula above. In yet another aspect, the natural or non-naturally occurring cysteine (C)-containing module comprises a polypeptide having at least five, six, or more disulfide bonds formed by pairing intra-protein cysteines according to a pattern selected from the group of permutations represented by the formula above. Any of the cysteine-containing proteins or scaffolds disclosed in the co-pending application Ser. Nos. 11/528,927 and 11/528,950, which are incorporated herein by reference in their entiety] are candidate binding modules.
- Binding modules can also be selected from libraries of cysteine-constrained cyclic peptides with 4, 5, 6, 7, 8, 9, 10, 11 and 12 randomized or partially randomized amino acids between the disulfide-bonded cystines (e.g., in a build-up manner), and in some cases additional randomized amino acids on the outside of the cystine pair can be constructed using a variety of methods. Library members with specificity for a target of interest can be identified using various methods including phage display, ribosomal display, yeast display and other methods known in the art. Such cyclic peptides can be utilized as binding modules in MURPs. In a preferred embodiment one can further engineer cysteine-constrained peptides to increase there binding affinity, proteolytic stability, and/or specificity using buildup approaches that lead to binding modules containing more than one disulfide bond. One particular buildup approach is illustrated in
FIG. 25 . It is based on the addition of a single cysteine plus multiple randomized residues on the N-terminal side of the previously selected cyclic peptide, as well as on the C-terminal side. One can generate libraries that have been designed as illustrated inFIG. 25 . Binding modules with improved properties can be identified by phage display or similar methods. Such buildup libraries can contain between 1 and 12 random positions on the N-terminal as well as on the C-terminal side of a cyclic peptide. The distance between the cysteine residues in the newly added random flanks and the cysteine residues in the cyclic peptide can be varied between 1 and 12 residues. Such libraries will contain four cysteine residues per library member, with two cysteines resulting from the original cyclic peptide and two cysteine residues in the newly added flanks. This approach favors a 1-4 2-3 DBP or a change in DBP, breaking up the preexisting 1-2 disulfide (=2-3 in the 4-cysteine construct) to form a 1-2 3-4 or a 1-3 2-4 DBP. Such buildup approaches can be performed with clone-specific primers so that it leaves no fixed sequence between the library areas as shown inFIG. 25 , or it can be performed with primers that use (and thus leave) a fixed sequence on both sides of the previously selected peptide and therefore these same primers can be used for any previously selected clone as illustrated inFIG. 26 . The method illustrated inFIG. 26 can be applied to a collection of cyclic peptides with specificity for a target of interest. Both buildup approaches were shown to work for anti-VEGF affinity maturation by build-up. This approach can be repeated to generate binding modules with six or more cysteine residues. - Another buildup of a one-disulfide into a 2-disulfide sequence is illustrated in
FIG. 27 . It involves the dimerization of a previously selected pool of 1-disulfide peptides with itself so that the preselected peptide pool ends up in the N-terminal as well as in the C-terminal position. This approach favors the build up of 2-disulfide sequences that recognize two separate epitopes on a target. - Another buildup approach involves the addition of a (partially) randomized sequence of 6-15 residues containing two cysteines that are spaced 4, 5, 6, 7, 8, 9, or 10 amino acids apart, with optionally additional randomized positions outside the linked cysteines. This 2-cysteine random sequence is added on the N-terminal side of the previously selected peptide, or on the C-terminal side. This approach favors a 1-2 3-4 DBP, although other DBPs may be formed. This approach can be repeated to generate binding modules with six or more cysteine residues.
- Binding modules can be constructed based on natural protein scaffolds. Such scaffolds can be identified by data base searching. Libraries that are based on natural scaffolds can be subjected to phage display panning followed by screening to identify sequences that specifically bind to a target of interest.
- A wide selection of natural scaffolds is available for constructing the binding modules. The choice of a particular scaffold will depend on the intended target. Non-limiting examples of natural scaffolds include snake-toxin-like proteins such as snake venom toxins and extracellular domain of human cell surface receptors. Non-limiting examples of snake venom toxins are Erabutoxin B, gamma-Cardiotoxin, Faciculin, Muscarininc toxin, Erabutoxin A, Neurotoxin I, Cardiotoxin V4II (Toxin III), Cardiotoxin V, alpha-Cobratoxin,
long Neurotoxin 1, FS2 toxin, Bungarotoxin, Bucandin, Cardiotoxin CTXI, Cardiotoxin CTX IIB, Cardiotoxin II, Cardiotoxin III, Cardiotoxin IV,Cobrotoxin 2, alpha-toxins, Neurotoxin II (cobrotoxin B), Toxin B (long neurotoxin), Candotoxin, Bucain. Non-limiting examples of extracellular domain of (human) cell surface receptors include CD59, Type II activin receptor, BMP receptor Ia ectodomain, TGF-beta type II receptor extracellular domain. Other natural scaffolds include but are not limited to A-domains, EGF, Ca-EGF, TNF-R, Notch, DSL, Trefoil, PD, TSP1, TSP2, TSP3, Anato, Integrin Beta, Thyroglobulin,Defensin 1,Defensin 2, Cyclotide, SHKT, Disintegrins, Myotoxins, Gamma-Thioneins, Conotoxin, Mu-Conotoxin, Omega-Atracotoxins, Delta-Atracotoxins, as well as additional families disclosed in co-pending application Ser. Nos. 11/528,927 and 11/528,950, which are incorporated herein in their entirety. - A large variety of methods has been described that allow one to identify binding molecules in a large library of variants. One method is chemical synthesis. Library members can be synthesized on beads such that each bead carries a different peptide sequence. Beads that carry ligands with a desirable specificity can be identified using labeled binding partners. Another approach is the generation of sub-libraries of peptides which allows one to identify specific binding sequences in an iterative procedure (Pinilla, C., et al. (1992) BioTechniques, 13: 901-905). More commonly used are display methods where a library of variants is expressed on the surface of a phage, protein, or cell. These methods have in common, that that DNA or RNA coding for each variant in the library is physically linked to the ligand. This enables one to detect or retrieve the ligand of interest and then determine its peptide sequence by sequencing the attached DNA or RNA. Display methods allow one skilled in the art to enrich library members with desirable binding properties from large libraries of random variants. Frequently, variants with desirable binding properties can be identified from enriched libraries by screening individual isolates from an enriched library for desirable properties. Examples of display methods are fusion to lac repressor (Cull, M., et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 1865-1869), cell surface display (Wittrup, K. D. (2001) Curr Opin Biotechnol, 12: 395-9). Of particular interest are methods were random peptides or proteins are linked to phage particles. Commonly used are M13 phage (Smith, G. P., et al. (1997) Chem Rev, 97: 391-410) and T7 phage (Danner, S., et al. (2001) Proc Natl Acad Sci USA, 98: 12954-9). There are multiple methods available to display peptides or proteins on M13 phage. In many cases, the library sequence is fused to the N-terminus of peptide pIII of the M13 phage. Phage typically carry 3-5 copies of this protein and thus phage in such a library will in most cases carry between 3-5 copies of a library member. This approach is referred to as multivalent display. An alternative is phagemid display where the library is encoded on a phagemid. Phage particles can be formed by infection of cells carrying a phagemid with a helper phage. (Lowman, H. B., et al. (1991) Biochemistry, 30: 10832-10838). This process typically leads to monovalent display. In some cases, monovalent display is preferred to obtain high affinity binders. In other cases multivalent display is preferred (O'Connell, D., et al. (2002) J Mol Biol, 321: 49-56).
- A variety of methods have been described to enrich sequences with desirable characteristics by phage display. One can immobilize a target of interest by binding to immunotubes, microtiter plates, magnetic beads, or other surfaces. Subsequently, a phage library is contacted with the immobilized target, phage that lack a binding ligand are washed away, and phage carrying a target specific ligand can be eluted by a variety of conditions. Elution can be performed by low pH, high pH, urea or other conditions that tend to break protein-protein contacts. Bound phage can also be eluted by adding E. coli cells such that eluting phage can directly infect the added E. coli host. An interesting protocol is the elution with protease which can degrade the phage-bound ligand or the immobilized target. Proteases can also be utilized as tools to enrich protease resistant phage-bound ligands. For instance, one can incubate a library of phage-bound ligands with one or more (human or mouse) proteases prior to panning on the target of interest. This process degrades and removes protease-labile ligands from the library (Kristensen, P., et al. (1998) Fold Des, 3: 321-8). Phage display libraries of ligands can also be enriched for binding to complex biological samples. Examples are the panning on immobilized cell membrane fractions (Tur, M. K., et al. (2003) Int J Mol Med, 11: 523-7), or entire cells (Rasmussen, U. B., et al. (2002) Cancer Gene Ther, 9: 606-12; Kelly, K. A., et al. (2003) Neoplasia, 5: 437-44). In some cases one has to optimize the panning conditions to improve the enrichment of cell specific binders from phage libraries (Watters, J. M., et al. (1997) Immunotechnology, 3: 21-9). Phage panning can also be performed in live patients or animals. This approach is of particular interest for the identification of ligands that bind to vascular targets (Arap, W., et al. (2002) Nat Med, 8: 121-7).
- A variety of cloning methods are available that allow one skilled in the art to generate libraries of DNA sequences that encode libraries of peptides. Random mixtures of nucleotides can be utilized to synthesize oligonucleotides that contain one or multiple random positions. This process allows one to control the number of random positions as well as the degree of randomization. In addition, one can obtain random or semi-random DNA sequences by partial digestion of DNA from biological samples. Random oligonucleotides can be used to construct libraries of plasmids or phage that are randomized in pre-defined locations. This can be done by PCR fusion as described in (de Kruif, J., et al. (1995) J Mol Biol, 248: 97-105). Other protocols are based on DNA ligation (Felici, F., et al. (1991) J Mol Biol, 222: 301-10; Kay, B. K., et al. (1993) Gene, 128: 59-65). Another commonly used approach is Kunkel mutagenesis where a mutagenized strand of a plasmid or phagemid is synthesized using single stranded cyclic DNA as template. See, Sidhu, S. S., et al. (2000) Methods Enzymol, 328: 333-63; Kunkel, T. A., et al. (1987) Methods Enzymol, 154: 367-82.
- Kunkel mutagenesis uses templates containing randomly incorporated uracil bases which can be obtained from E. coli strains like CJ236. The uracil-containing template strand is preferentially degraded upon transformation into E. coli while the in vitro synthesized mutagenized strand is retained. As a result most transformed cells carry the mutagenized version of the phagemid or phage. A valuable approach to increase diversity in a library is to combine multiple sub-libraries. These sub-libraries can be generated by any of the methods described above and they can be based on the same or on different scaffolds.
- A useful method to generate large phage libraries of short peptides has been recently described (Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51). This method is related to the Kunkel approach but it does not require the generation of single stranded template DNA that contains random uracil bases. Instead, the method starts with a template phage that carries one or more mutations close to the area to be mutagenized and said mutation renders the phage non-infective. The method uses a mutagenic oligonucleotide that carries randomized codons in some positions and that correct the phage-inactivating mutation in the template. As a result, only mutagenized phage particles are infective after transformation and very few parent phage are contained in such libraries. This method can be further modified in several ways. For instance, one can utilize multiple mutagenic oligonucleotides to simultaneously mutagenize multiple discontiguous regions of a phage. We have taken this approach one step further by applying it to whole microproteins of >25, 30, 35, 40, 45, 50, 55 and 60 amino acids, instead of short peptides of <10, 15 or 20 amino acids, which poses an additional challenge. This approach now yields libraries of more than 10e10 transformants (up to 10e11) with a single transformation, so that a single library with a diversity of 10e12 is expected from 10 transformations.
- Another variation of the Scholle method is to design the mutagenic oligonucleotide such that an amber stop codon in the template is converted into an ochre stop codon, and an ochre into an amber in the next cycle of mutagenesis. In this case the template phage and the mutagenized library members must be cultured in different suppressor strains of E. coli, alternating an ochre suppressor with amber suppressor strains. This allows one to perform successive rounds of mutagenesis of a phage by alternating between these two types of stop codons and two suppressor strains.
- Yet another variation of the Scholle approach involves the use of megaprimers with a single stranded phage DNA template. The megaprimer is a long ssDNA that was generated from the library inserts of the selected pool of phage from the previous round of panning. The goal is to capture the full diversity of library inserts from the previous pool, which was mutagenized in one or more areas, and transfer it to a new library in such a way that an additional area can be mutagenized. The megaprimer process can be repeated for multiple cycles using the same template which contains a stop-codon in the gene of interest. The megaprimer is a ssDNA (optionally generated by PCR) which contains 1) 5′ and 3′ overlap areas of at least 15 bases for complementarity to the ssDNA template, and 2) one or more previously selected library areas (1, 2, 3, 4 or more) which were copied (optionally by PCR) from the pool of previously selected clones, and 3) a newly mutagenized library area that is to be selected in the next round of panning. The megaprimer is optionally prepared by 1) synthesizing one or more oligonucleotides encoding the newly synthesized library area and 2) by fusing this, optionally using overlap PCR, to a DNA fragment (optionally obtained by PCR) which contains any other library areas which were previously optimized. Run-off or single stranded PCR of the combined (overlap) PCR product is used to generate the single stranded megaprimer that contains all of the previously optimized areas as well as the new library for an additional area that is to be optimized in the next panning experiment. This approach is expected to allow affinity maturation of proteins using multiple rapid cycles of library creation generating 10e11 to 10e12 diversity per cycle, each followed by panning.
- A variety of methods can be applied to introduce sequence diversity into (previously selected or naïve) libraries of microproteins or to mutate individual microprotein clones with the goal of enhancing their binding or other properties like manufacturing, stability or immunogenicity. In principle, all the methods that can be used to generate libraries can also be used to introduce diversity into enriched (previously selected) libraries of microproteins. In particular, one can synthesize variants with desirable binding or other properties and design partially randomized oligonucleotides based on these sequences. This process allows one to control the positions and degree of randomization. One can deduce the utility of individual mutations in a protein from sequence data of multiple variants using a variety of computer algorithms (Jonsson, J., et al. (1993) Nucleic Acids Res, 21: 733-9; Amin, N., et al. (2004) Protein Eng Des Sel, 17: 787-93). Of particular interest for the re-mutagenesis of enriched libraries is DNA shuffling (Stemmer, W. P. C. (1994) Nature, 370: 389-391), which generates recombinants of individual sequences in an enriched library. Shuffling can be performed using a variety modified PCR conditions and templates may be partially degraded to enhance recombination. An alternative is the recombination at pre-defined positions using restriction enzyme-based cloning. Of particular interest are methods utilizing type IIS restriction enzymes that cleave DNA outside of their sequence recognition site (Collins, J., et al. (2001) J Biotechnol, 74: 317-38. Restriction enzymes that generate non-palindromic overhangs can be utilized to cleave plasmids or other DNA encoding variant mixtures in multiple locations and complete plasmids can be re-assembled by ligation (Berger, S. L., et al. (1993) Anal Biochem, 214: 571-9). Another method to introduce diversity is PCR-mutagenesis where DNA sequences encoding library members are subjected to PCR under mutagenic conditions. PCR conditions have been described that lead to mutations at relatively high mutation frequencies (Leung, D., et al. (1989) Technique, 1: 11-15). In addition, a polymerase with reduced fidelity can be employed (Vanhercke, T., et al. (2005) Anal Biochem, 339: 9-14). A method of particular interest is based on mutator strains (Irving, R. A., et al. (1996) Immunotechnology, 2: 127-43; Coia, G., et al. (1997) Gene, 201: 203-9). These are strains that carry defects in one or more DNA repair genes. Plasmids or phage or other DNA in these strains accumulate mutations during normal replication. One can propagate individual clones or enriched populations in mutator strains to introduce genetic diversity. Many of the methods described above can be utilized in an iterative process. One can apply multiple rounds of mutagenesis and screening or panning to entire genes, or to portions of a gene, or one can mutagenize different portions of a protein during each subsequent round (Yang, W. P., et al. (1995) J Mol Biol, 254: 392-403).
- The libraries can be further treated to reduce artifacts. Known artifacts of phage panning include 1) no-specific binding based on hydrophobicity, and 2) multivalent binding to the target, either due to a) the pentavalency of the pIII phage protein, or b) due to the formation of disulfides between different microproteins, resulting in multimers, or c) due to high density coating of the target on a solid support and 3) context-dependent target binding, in which the context of the target or the context of the microproteins becomes critical to the binding or inhibition activity. Different treatment steps can be taken to minimize the magnitude of these problems. For example, such treatments are applied to the whole library, but some useful treatments that remove bad clones can only be applied to pools of soluble proteins or only to individual soluble proteins.
- Libraries of cysteine-containing scaffolds are likely to contain free thiols, which can complicate directed evolution by cross-linking to other proteins. One approach is to remove the worst clones from the library by passing it over a free-thiol column, thus removing all clones that have one or more free sulfhydryls. Clones with free SH groups can also be reacted with biotin-SH reagents, enabling efficient removal of clones with reactive SH groups using Streptavidin columns. Another approach is to not remove the free thiols, but to inactivate them by capping them with sulfhydryl-reactive chemicals such as iodoacetic acid. Of particular interest are bulky or hydrophilic sulfhydryl reagents that reduce the non-specific target binding or modified variants.
- Examples of context dependence are all of the constant sequences, including pIII protein, linkers, peptide tags, biotin-streptavidin, Fc and other fusion proteins that contribute to the interaction. The typical approach for avoiding context-dependence involves switching the context as frequently as practical in order to avoid buildup. This may involve alternating between different display systems (ie M13 versus T7, or M13 versus Yeast), alternating the tags and linkers that are used, alternating the (solid) support used for immobilization (ie immobilization chemistry) and alternating the target proteins itself (different vendors, different fusion versions).
- Library treatments can also be used to select for proteins with preferred qualities. One option is the treatment of libraries with proteases in order to remove unstable variants from the library. The proteases used are typically those that would be encountered in the application. For pulmonary delivery, one would use lung proteases, for example obtained by a pulmonary lavage. Similarly, one would obtain mixtures of proteases from serum, saliva, stomach, intestine, skin, nose, etc. However, it is also possible to use mixtures of single purified proteases. An extensive list of proteases is shown in [Appendix E]. The phage themselves are exceptionally resistant to most proteases and other harsh treatments.
- For example, it is possible to select the library for the most stable structures, ie those with the strongest disulfide bonds, by exposing it to increasing concentrations of reducing agents (ie DTT or betamercaptoethanol), thus eliminating the least stable structures first. One would typically use reducing agent (ie DTT, BME, other) concentrations from 2.5 mM, to 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or even 100 mM, depending on the desired stability.
- It is also possible to select for clones that can be efficiently refolded in vitro, by reducing the entire display library with a high level of reducing agent, followed by gradually re-oxidizing the protein library to reform the disulfides, followed by the removal of clones with free SH groups, as described above. This process can be applied once or multiple times to eliminate clones that have low refolding efficiency in vitro.
- One approach is to apply a genetic selection for protein expression level, folding and solubility as described by A. C. Fisher et al. (2006) Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway. Protein Science (online). After panning of display libraries (optional), one would like to avoid screening thousands of clones at the protein level for target binding, expression level and folding. An alternative is to clone the whole pool of selected inserts into a betalactamase fusion vector, which, when plated on betalactam, the authors demonstrated to be selective for well-expressed, fully disulfide bonded and soluble proteins.
- Following M13 Phage display of protein libraries and panning on targets for one or more cycles, there are a variety of ways to proceed, including (1) screening of individual phage clones by phage ELISA, which measures the number of phage particles (using anti-M13 antibodies) that bind to an immobilized target; (2) transferring from M13 into T7 phage display libraries. The second approach is particularly useful in reducing the occurrence of false positives based on valency. Any single library format tends to favor clones that can form high-avidity contacts with the target. This is the reason that screening of soluble proteins is important, although this is a tedious solution. The multivalency achieved in T7 phage display is likely very different from that achieved in M13 display, and cycling between T7 and M13 can be an excellent approach to reducing the occurrence of false positives based on valency.
- Filter lift is another methodology that can be with bacterial colonies grown at high density on large agar plates (10e2-10e5). Small amounts of some proteins are secreted into the media and end up bound to the filter membrane (nitrocellulose or nylon). The filters are then blocked in non-fat milk, 1% Casein hydrolysate or a 1% BSA solution and incubated with the target protein that has been labeled with a fluorescent dye or an indicator enzyme (directly or indirectly via antibodies or via biotin-streptavidin). The location of the colony is determined by overlaying the filter on the back of the plate and all of the positive colonies are selected and used for additional characterization. The advantage of filter lifts is that it can be made to be affinity-selective by reading the signal after washing for different periods of time. The signal of high affinity clones ‘fades’ slowly, whereas the signal of low affinity clones fades rapidly. Such affinity characterization typically requires a 3-point assay with a well-based assay and may provide better clone-to-clone comparability than well-based assays. Gridding of colonies into an array is useful since it minimizes differences due to colony size or location.
- N-Terminal Modules:
- The subject MURPs can contain N-terminal modules (NM), which are particularly useful e.g., in facilitating production of the MURPs. The NM can be a single methionine residue when the products is expressed in the E. coli cytoplasm. A typical product format is an URP fused to a therapeutic protein, which is expressed in the bacterial cytoplasm so that the N-terminus is formyl-methionine. The formyl-methionine can either be permanent or temporary, if it is removed by biological or chemical processing.
- The NM can also be a peptide sequence that has been engineered for proteolytic processing, which can be used to remove tags or to remove fusion proteins. The N-terminal module can be engineered to facilitate the purification of the MURP by including an affinity tag such as the Flag-, Myc-, HA- or His-tag. The N-terminal module can also include an affinity tag that can be used for the detection of the MURP. An NM can be engineered or selected for high-level expression of the MURP. It can also be engineered or selected to enhance the protease resistance of the resulting MURP. MURPs can be produced with an N-terminal module that facilitates expression and/or purification. This N-terminal module can be cleaved off during the production process with a protease, such that the final product does not contain an N-terminal module.
- By optimizing the amino acid and codon choice of the N-terminal module one can increase recombinant production. The N-terminal module can also contain a processing site that can be cleaved by a specific protease like factor Xa, thrombin, or enterokinase, Tomato Etch Virus (TEV) protease. Processing sites can also be designed to be cleavable by chemical hydrolysis. An example is the amino acid sequence asp-pro that can be cleaved under acidic conditions. An N-terminal module can also be designed to facilitate the purification of a MURP. For example, N-terminal modules can be designed to contain multiple his residues which allow product capture by immobilized metal chromatography. N-terminal modules can contain peptide sequences that can be specifically captured or detected by antibodies. Examples are FLAG, HA, c-myc.
- C-Terminal Modules:
- MURPs can contain a C-terminal module, which are particularly useful e.g., in facilitating production of the MURPs. For example, C-terminal module can comprise a cleavage site to effect proteolytic processing to remove sequences that are fused and hence increasing protein expression or facilitating purification. In particular, the C-terminal module can also contain a processing site that can be cleaved by a specific protease like factor Xa, thrombin, TEV protease or enterokinase. Processing sites can also be designed to be cleavable by chemical hydrolysis. An example is the amino acid sequence asp-pro that can be cleaved under acidic conditions. The C-terminal module can be an affinity tag aimed at facilitating the purification of the MURP. For example, C-terminal modules can be designed to contain multiple his residues which allow product capture by immobilized metal chromatography. C-terminal modules can contain peptide sequences that can be specifically captured or detected by antibodies. Non-limiting examples of the tags include FLAG-, HA-, c-myc, or His-tag. C-terminal module can also be engineered or selected to enhance the protease resistance of the resulting MURP.
- Where desired, the N-terminus of the protein can be linked to its own C-terminus. For example, linking these two modules can be carried out by creating an amino acid-like natural linkage (peptide bond) or by using an exogenous linking entity. Of particular interest are cyclotides, a family of small proteins in which this occurs naturally. Adopting a structural format like cyclotides is expected to provide additional stability against exo-proteases. Such intramolecular linkage typically works better at lower protein concentrations.
- Effector Modules:
- MURPs can comprise one or multiple effector modules (EMs), or none at all. Effector modules typically do not provide the targeting, but they provide an activity required for therapeutic effect, like cell-killing. EMs can be pharmaceutically active small molecules (ie toxic drugs), peptides or proteins. Non-limiting examples are cytokines, antibodies enzymes, growth factors, hormones, receptors, receptor agonists or antagonists, whether whole or a fragment or domain thereof. Effector modules can also comprise peptide sequences that carry chemically linked small molecule drugs, whether synthetic or natural. Optionally, these effector molecules can be linked to the effector module via chemical linkers, which may or may not be cleaved under selected conditions leading to a release of the toxic activity. EMs can also include radioisotopes and their chelates, as well as various labels for PET and MRI. Effector modules can also be toxic to a cell or a tissue. Of particular interest are MURPs that contain toxic effector modules and binding modules with specificity for a diseased tissue or disease cell type. Such MURPs can specifically accumulate in a diseased tissue or in diseased cells and the can exert their toxic action preferentially in the diseased cells or tissues. Listed below are exemplary effector modules.
- Enzymes—Effector modules can be enzymes. Of particular interest are enzymes that degrade metabolites that are critical for cellular growth like carbohydrates or amino acids or lipids or co-factors. Other examples for effector modules with enzymatic activity are RNase, DNase, and phosphatase, asparaginase, histidinase, arginase, betalactamase. Effector modules with enzymatic activity can be toxic when delivered to a tissue or cell. Of particular interest are MURPs that combine effector modules that are toxic and binding modules that bind specifically to a diseased tissue. Enzymes that convert an inactive prodrug into an active drug at the tumor site are also potential effector modules.
- Drug—The subject MURP can contain an effector that is a drug. Where desired, sequences can be designed for the organ-selective delivery of drug molecules. An example is illustrated in
FIG. 8 . An URP sequence can be fused to a protein that preferentially binds to diseased tissue. The same URP sequence can contain one or more amino acid residues that can be modified for the attachment of drug molecules. Such a conjugate can bind to diseased tissue with high specificity and the attached drug molecules can result in local action while minimizing systemic drug exposure. The MURP can be designed to facilitate the release of drug molecules at the target size by introducing protease-sensitive sites that can be cleaved by native proteases at the site of desired action. A significant advantage of using URP sequences for the design of drug delivery constructs is that one can avoid undesirable interactions between the drug molecule and the targeting domain of the construct. Many drug molecules that can be conjugated to targeting domains have significant hydrophobicity and the resulting conjugates tend to aggregate. By adding hydrophilic URP sequences to such constructs one can improve the solubility of the resulting delivery constructs and as a consequence reduce the aggregation tendency. Furthermore, one can increase the number of drug molecules that can be fused to a targeting domain by adding long URP sequences. In addition, the use of URP sequences allows one to optimize the distance between the drug conjugation sites to facilitate complete conjugation. The list of suitable drugs includes but are not limited to chemotherapeutic agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; duocarmycin, maytansin, auristatin, elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.R™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethyla-mine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-1); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin, doxorubicin, daunomycin, duocarmycin, vincristin, and vinblastin. - Other drugs that can be used as the effector modules include those that are useful for treating inflammatory conditions, cardiac diseases, infectious diseases, respiratory diseases, autoimmune diseases, neronal and muscular disorders, metabolic disorders, and cancers.
- Additional drugs that can be used as the effectors in MURPs include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are generated by biotransformation of the products of the selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory agents, analgesic-antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of the inducible cyclooxygenase, selective inhibitors of the inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, β-adrenergic agonists, ipratropium, glucocorticoids, methylxanthines, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers and leukotriene inhibitors.
- Other drugs that can be used as effector include agents for the treatment of peptic ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetics, agents used in irritable bowel syndrome, agents used for diarrhea, agents used for constipation, agents used for inflammatory bowel disease, agents used for biliary disease, agents used for pancreatic disease.
- Radionuclides—MURPs can be designed for the tissue-targeted delivery of radionuclides as well as for imagin with radionuclides. URPs are ideal for imaging because the halflife can be optimized by changing the length of the URP. For most imaging applications a moderately long URP is likely to be preferred, providing a halflife of 5 minutes to a few hours, not days or weeks MURPs can be designed such that they only contain a single or a small defined number of amino groups that can be modified with chelating agents (such as DOTA) for radio isotopes such as technetium, indium, yttrium, (EXPAND). Alternative methods of conjugation are through reserved cysteine side chains. Such radionuclide-carrying MURPs can be employed for the treatment of tumors or other diseased tissues, as well as for imaging.
- Many pharmaceutically active proteins or protein domains can used as effector models in MURPs. Examples are the following proteins as well as fragments of these proteins: cytokines, growth factors, enzymes, -receptors, microproteins, hormones, erythopoetin, adenosine deiminase, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, rasburicase, axokine, RNAse, DNAse, phosphatase, pseudomonas exotoxin, ricin, gelonin, desmoteplase, laronidase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor IIV, factor VIII, factor X, factor IX, dornase, glucocerebrosidase, follitropin, glucagon, thyrotropin, nesiritide, alteplase, teriparatide, agalsidase, laronidase, methioninase.
- Protease-activated MURPs: To enhance the therapeutic index of an effector module, one can insert protease-labile sequences into URP sequences that are sensitive to proteases that are preferentially found in serum or in the target tissue to be treated by the MURP. This approach is illustrated in
FIG. 9 . Some designs allows one to construct proteins that are selectively activated when reaching a target tissue. Of particular interest are MURPs that are activated at a disease site. To facilitate such target-specific activation one can attach URP sequences in close proximity to the active site or receptor binding site of the effector module such that the resulting fusion protein has limited biological activity. Of particular interest is the activation of an effector module at a tumor site. Many tumor tissues express proteases in relatively high concentrations and sequences that are specifically cleaved by these tumor proteases can be inserted into URP sequences. For example, most prostate tumor tissues contain high concentrations of prostate specific antigen (PSA) which is a serine protease. Prodrugs consisting of a PSA-labile peptide conjugated to the cancer drug doxorubicin have shown selective activation in prostate tissue [DeFeo-Jones, D., et al. (2000) Nat Med, 6: 1248]. Of particular interest for disease-specific activation are proteins with cytostatic or cytotoxic activity like TNFalpha, and many cytokines and interleukins. Another application is the selective activation of proteins at the site of inflammation or at site of virus or bacterial infection. - Methods of production—MURPs containing URP sequences can be produced using molecular biology approaches that are well know in the art. A variety of cloning vectors are available for various expression systems like mammalian cells, yeast, and microbes. Of particular interest as expression hosts are E. coli, S. cerevisiae, P. pastoris, and chinese hamster ovary cells. Of particular interest are hosts that have been optimized to widen their codon usage. Of particular interest is a host that has been modified to enhance expression of GRS. That can be done by providing DNA that encodes glycine-specific tRNAs. In addition, one can engineer the host such that loading of glycine-specific tRNAs is enhanced. The DNA encoding the enhanced protein can be operationally linked to a promoter sequences. The DNA encoding the enhanced protein as well as the operationally linked promoter can be part of a plasmid vector, viral vector or it can be inserted into the chromosome of the host.
- For production on can culture the host under conditions that facilitate the production of the enhanced protein. Of particular interest are conditions that improve the production of GRS.
- The subject MURPs can adopt a variety of formats. For instance, the MURPs can contain URPs that are fused to pharmaceutically active proteins to produce slow-release products. Such products can be injected or implanted locally for instance into or under the skin of a patient. Due to its large hydrodynamic radius the URP sequences-containing product is slowly released from the injection or implantation site which leads to a reduction of the frequency of injection or implantation. The URP sequences can be designed to contain regions that bind to cell surfaces or tissue in order to prolong the local retention of the drug at the injection site. Of particular interest are URP-containing products that can be formulated as soluble compounds but form aggregates or precipitates upon injection. This aggregation or precipitation can be triggered by a change in pH between the formulated product and the pH at the injection site. Alternatives are URP-containing products that precipitate or form aggregates as a result of a change in redox conditions. Yet another approach is a URP-containing product that is stabilized in solution by addition of non-active solutes, but that precipitates or aggregates upon injection as a result of diffusion of the solubilizing solutes. Another approach is to design URP-containing products that contain one or multiple Lysine or Cysteine residues in their URP sequence and that can be cross-linked prior to injection.
- Where desired, the MURP is monomeric (here meaning not-crosslinked) when manufactured and formulated and when injected, but after subcutaneous injection the protein starts to crosslink with itself or with native human proteins, forming a polymer under the skin from which active drug molecules are freed only very gradually. Such release can be by disulfide bond reduction or disulfide shuffling as illustrated in
FIG. 18 , or it can be mediated by proteolysis as shown inFIG. 19 , releasing active fragments into the circulation. It is important that these active fragments are large enough to have a long halflife, because the longer their secretion halflife, the lower the dose of the released protein can be, allowing the use of a lower dose of product to be injected or a longer time between injections. - One approach that offers these advantages is disulfide-mediated crosslinking of proteins. For example, a protein drug would be manufactured with a cyclic peptide in it (one or more). This cyclic peptide may or may not be involved in binding to the target. This protein is manufactured with the cyclic peptide formed, ie in oxidized form, to simplify purification. However, the product is then reduced and formulated to keep the protein in reduced form. It is important that the cyclic peptide reduces at a low concentration of reducing agent, such as 0.25, 0.5, 1.0, 2.0, 4.0 or 8.0 mM Dithiothreitol or Betamercaptoethanol or cysteine or equivalent reducing agent, so that the cyclic peptide can be reduced without reducing other disulfide containing protein modules in the product. The use of FDA approved reducing agents is preferred, such as cysteine or glutathione. After subcutaneous injection, the low molecular weight reducing agent diffuses away rapidly or is neutralized by human proteins, exposing the drug to an oxidizing environment while it is still at a high molar concentration, which causes crosslinking of cysteines located on different protein chains, which leads to polymerization of the drug at the injection site. The longer the distance between the cysteines in the cyclic peptide, and the higher the concentration of the drug, the higher the degree of polymerization of the drug will be, since polymerization competes with cyclic peptide reformation. Over time, disulfide reduction and oxidation will cause disulfide reshuffling, which will lead to cyclic peptide reformation and monomerization and resolubilization of the drug. The release of the drug from the polymer can also occur via proteolysis which could be targeted and controlled or increased by building in cleavage sites for serum proteases. The crosslinking to the proteins could also be performed with a chemical protein-protein crosslinking agent, such as the ones listed in [table x]. Ideally, this is an already FDA-approved agent, such as those used for vaccine conjugation or conjugation of chemicals to proteins.
- Instead of using disulfides, one can also stabilize proteins against proteolytic degradation using a wide variety of crosslinking agents. Most of the agents below are sold by Pierce Chemicals under that same name and instructions for their use are available online (www.piercenet.com). The agents that result in the same chain-to-chain distance as obtained with disulfides are the most likely to be useful for this application. The short-linker agents such as DFDNB are the most promising. The interchain distance can be readily determined from the structures of the chemicals as shown in www.piercenet.com.
- There are a large number of specific chemical products that work based on the following small number of basic reaction schemes, all of which are described in detail at www.piercenet.com. Examples of useful crosslinking agents are Imidoesters, active halogens, maleimide, pyridyl disulfide, NHS-ester. Homobifunctional crosslinking agents have two identical reactive groups and are often used in a onestep chemical crosslinking procedure. Examples are BS3 (a non-cleavable water-soluble DSS analog), BSOCOES (base-reversible), DMA (Dimethyl adipimidate-2HCl), DMP (Dimethyl pimelimidate-2HCl), DMS (Dimethyl suberimidate-2HCl), DSG (5-carbon analog of DSS), DSP (Lomant's reagent), DSS (non-cleavable), DST (cleavable by oxidizing agents), DTBP (
Dimethyl - Sulfhydryl-reactive homobifunctional crosslinking agents are homobifunctional protein crosslinkers that react with sulfhydryls are often based on maleimides, which react with —SH groups at pH 6.5-7.5, forming stable thioether linkages. BM[PEO]3 is an 8-atom polyether spacer that reduces potential for conjugate precipitation in sulfhydryl-to-sulfhydryl cross-linking applications. BM[PEO]4 is similar but with an 11-atom spacer. BMB is a non-cleavable crosslinker with a four-carbon spacer. BMDB makes a linkage that can be cleaved with periodate. BMH is a widely used homobifunctional sulfhydryl-reactive crosslinker. BMOE has an especially short linker. DPDPB and DTME are cleavable crosslinkers. HVBS does not have the hydrolysis potential of meleimides. TMEA is another option. Hetero-bifunctional crosslinking agents have two different reactive groups. Examples are NHS-esters and amines/hydrazines via EDC activation, AEDP, ASBA (photoreactive, iodinatable), EDC (water-soluble carbodiimide). Amine-Sulflhydryl reactive bifunctional crosslinkers are AMAS, APDP, BMPS, EMCA, EMCS, GMBS, KMUA, LC-SMCC, LC-SPDP, MBS, SBAP, SIA (extra short), SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-LC-SMPT, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB. Amino-group reactive heterobifunctional crosslinking agents are ANB-NOS, MSA, NHS-ASA, SADP, SAED, SAND, SANPAH, SASD, SFAD, Sulfo-HSAB, Sulfo-NHS-LC-ASA, Sulfo-SADP, Sulfo-SANPAH, TFCS.
- A different slow release format has the drug labeled with a His6 tag, which is mixed and co-injected with Nickel-Nitrilotriacetic acid-conjugated beads (Ni-NTA beads), a GMO version of the ones that are available from Qiagen. The drug would slowly teach off the beads, providing depot and slow release as illustrated in
FIG. 20 . The beads are optional and can be replaced by a crosslinked, polymeric Nickel-nitrilotriacetic acid that leads to assembly of an even larger polymer. - URP sequences can contain sequences that are known to form multimers like alpha2D [Hill, R., et al. (1998) J Am Chem Soc, 120: 1138-1145] that was utilized to dimerize an antibody fragment [Kubetzko, S., et al. (2005) Mol Pharmacol, 68: 1439-54]. Examples of a useful homo dimerization peptide is the sequence SKVILFE. An example of useful heterodimerization sequences are the peptide ARARAR that can form dimers with the sequence DADADA and related sequences. Multimerization can improve the biological function of a molecule by increasing its avidity and it can influence pharmacokinetic properties and tissue distribution of the resulting MURPs.
- “Multimerization modules” are amino acid sequences that facilitate dimer or multimer formation of MURPs. Multimerization modules may bind to themselves to form dimers or multimers. Alternatively, multimerization modules can bind to other modules of the MURP. These can be leucine zippers or small peptides like Hydra head activator derivatives (SKVILF-like) which forms antiparallel homopolymers, or peptides like RARARA and DADADA, which form high affinity antiparallel heteropolymers. Using one, two or more copies of these peptides one can force the formation of protein dimers, linear multimers or branched multimers.
- The affinity of the association can be tailored by changing the type, length and composition of the peptides. Some applications require peptides that form homodimers as illustrated in
FIG. 21 . Other applications require heterodimers. In some cases, once associated, the peptides can be locked into place by forming disulfide bonds between the two protein chains, typically on either side of the peptides. Multimerization modules are useful for linking two MURP molecules together (head to tail, head to head, or tail to tail) as illustrated inFIG. 21 . The multimerization modules can be located on either the N- or C-terminus in order to form dimers. If the multimerization modules are present at both termini, long, linear multimers will be formed. If more than two multimerization modules are present per protein, branched polymeric networks can be formed. The concepts of multimerization and chemical conjugation can be combined leading to useful for halflife extension and depot formation, leading to slow release of active drug from the depot or injection site as illustrated inFIG. 23 . - The subject MURPs can incorporate a genetic or universal URP. One approach is to express a URP containing a long URP module, which provides halflife and contains multiple (typically 4-10) lysines (or other sites) that allows site-specific conjugation of peptides (ie linear, cyclic, 2SS, 3 SS, etc) that bind to a specific target. The advantage of this approach is that the URP module is generic and can be conjugated with any target-specific peptide. Ideally the linkage of the target-specific peptide to the URP is a directed linkage, so that residues on the URP can only react with a residue on the target-specific peptide and exhaustive coupling can only produce a single species, which is a URP that is linked to a peptide at every lysine, for example. This complex behaves like a high-avidity multimer in it's binding properties but is simple to manufacture. This approach is illustrated in
FIG. 24 . - The subject MURPs can also incorporate URPs to effect delivery across tissue barriers. URPs can be engineered to enhance delivery across the dermal, oral, buccal, intestinal, nasal, blood-brain, pulmonary, thecal, peritoneal, rectal, vaginal or many other tissue barriers.
- One of the key obstacles to oral protein delivery is the sensitivity of most proteins to proteases in the digestive system. Conjugation to URP sequences can improve protease resistance of pharmaceutically active proteins and thus facilitate their uptake. It has been shown that protein uptake in the digestive system can be improved by adding molecular carriers. The main role of these carriers is an improvement of membrane permeability [Stoll, B. R., et al. (2000) J Control Release, 64: 217-28]. Thus one can include sequences into URP sequences that improve membrane permeability. Many sequences that improve membrane permeability are know and examples are sequences rich in arginine [Takenobu, T., et al. (2002) Mol Cancer Ther, 1: 1043-9]. Thus one can design URP sequences that improve cellular or oral uptake of proteins by combining two functions, a reduction in proteolytic degradation of the protein of interest as well as an increase in membrane permeability of the fusion product. Optional, on can add a sequence to the URP sequence that is sensitive to a protease that is preferentially located at in the target tissue for the drug of interest but is stable to proteases in the digestive tract. Examples of such URP sequences are sequences that contain long regions of GRS as well as sequences that are rich in basic amino acids in particular arginine and facilitate membrane transfer. URP can be utilized in a similar way to improve protein uptake via intranasal, intrapulmonary, or other routes of delivery.
- DR4/DR5 agonist—DR4 and DR5 are death receptors that are expressed on many tumor cells. These receptors can be triggered by trimerization which leads to cell death and tumor regression. Binding domains with specificity for DR4 or DR5 can be obtained by phage panning or other display methods. These DR4 or DR5-specific binding domains can be multimerized using URP modules as linkers as illustrated in
FIG. 12 . Of particular interest are MURPs that contain three or more binding modules with specificity for DR4 or DR5 or both. As illustrated inFIG. 12 , MURPs can contain additional binding modules with specificity for tumor antigens that are overexpressed in tumor tissues. This allows one to construct MURPs that specifically accumulate in tumor tissue and trigger cell death. MURPs can contain modules that bind either DR4 or DR5. Of particular interest are MURPs that contain binding modules that bind both DR4 and DR5. - Tumor-targeted
Interleukin 2—Interleukin 2 (IL2) is a cytokine that can enhance the immune response to tumor tissue. However, systemic IL2 therapy is characterized by significant side effects. MURPs can be constructed that combine binding domains with specificity for tumor antigens and IL2 as effector module as illustrated inFIG. 13 . Such MURP can selectively accumulate in tumor tissue and thus elicit a tumor-selective immune response while minimizing the systemic side effects of cytokine therapy. Such MURPs can target a variety of tumor antigens like EpCAM, Her2, CEA, EGFR, Thomsen Friedenreich antigen. Of particular utility are MURPs that bind to tumor antigens that show slow internalization. Similar MURPs can be designed using other cytokines or tumor necrosis factor-alfa as effector modules. - Tumor-selective asparaginase—Asparaginase is used to treat patients with acute leukemia. Both asparaginase from E. coli and asparaginase from Erwinia are used for treatment. Both enzymes can lead to immunogenicity and hypersensitive reactions. Oncaspar is PEGylated version of asparaginase that has reduced immunogenicity. However, the protein is difficult to manufacture and administered as a mixture of isomers. Adding URP sequences to termini and/or to internal loops allows the direct recombinant manufacture of an asparaginase variant that is homogeneous and has low immunogenicity. Various URP sequences and attachment sites can be compared to determine the optimum position for URP sequence attachment. Several other enzymes can degrade amino acids have reported antitumor activity. Examples are arginase, methioninase, phenylalanine ammonia lyase, and tryptophanase. Of particular interest is the phenylalanine ammonia lyase of streptomyces maritimus, which has a high specific activity and does not require a co-factor [Calabrese, J. C., et al. (2004) Biochemistry, 43: 11403-16]. Most of these enzymes are of bacterial or other non-human origin and are likely to elicit immune reactions. The immunogenicity of these enzymes can be reduced by adding one or more URP sequences. In addition, the therapeutic index and PK properties of these enzymes can be improved by increasing their hydrodynamic radius as a result of URP sequences attachment.
- The subject MURPs can be designed to target any cellular proteins. A non-limiting list is provided below.
- VEGF, VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, EGF-1, EGF-2, EGF-3, Alpha3, cMet, ICOS, CD40L, LFA-1, c-Met, ICOS, LFA-1, IL-6, B7.1, B7.2, OX40, IL-1b, TACI, IgE, BAFF or BLys, TPO-R, CD19, CD20, CD22, CD33, CD28, IL-1-R1, TNFα, TRAIL-R1, Complement Receptor 1, FGFa, Osteopontin, Vitronectin, Ephrin A1-A5, Ephrin B1-B3, alpha-2-macroglobulin, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CCL13, CCL14, CCL15, CXCL16, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, PDGF, TGFb, GMCSF, SCF, p40 (IL12/IL23), IL1b, IL1a, IL1ra, IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL23, Fas, FasL, Flt3 ligand, 41BB, ACE, ACE-2, KGF, FGF-7, SCF, Netrin1,2, IFNa,b,g, Caspase2,3,7,8,10, ADAM S1,S5,8,9,15,TS1,TS5; Adiponectin, ALCAM, ALK-1, APRIL, Annexin V, Angiogenin, Amphiregulin, Angiopoietin1,2,4, B7-1/CD80, B7-2/CD86, B7-H1, B7-H2, B7-H3, Bcl-2, BACE-1, BAK, BCAM, BDNF, bNGF, bECGF, BMP2,3,4,5,6,7,8; CRP, Cadherin6,8,11; Cathepsin A,B,C,D,E,L,S,V,X; CD11a/LFA-1, LFA-3, GP2b3a, GH receptor, RSV F protein, IL-23 (p40, p19), IL-12, CD80, CD86, CD28, CTLA-4, α4β1, α4β7, TNF/Lymphotoxin, IgE, CD3, CD20, IL-6, IL-6R, BLYS/BAFF, IL-2R, HER2, EGFR, CD33, CD52, Digoxin, Rho (D), Varicella, Hepatitis, CMV, Tetanus, Vaccinia, Antivenom, Botulinum, Trail-R1, Trail-R2, cMet, TNF-R family, such as LA NGF-R, CD27, CD30, CD40, CD95, Lymphotoxin a/b receptor, Wsl-1, TL1A/TNFSF15, BAFF, BAFF-R/TNFRSF13C, TRAIL R2/TNFRSF10B, TRAIL R2/TNFRSF10B, Fas/TNFRSF6 CD27/TNFRSF7, DR3/TNFRSF25, HVEM/TNFRSF14, TROY/TNFRSF19, CD40 Ligand/TNFSF5, BCMA/TNFRSF17, CD30/TNFRSF8, LIGHT/TNFSF14, 4-1BB/TNFRSF9, CD40/TNFRSF5, GITR/TNFRSF18, Osteoprotegerin/TNFRSF11B, RANK/TNFRSF11A, TRAIL R3/TNFRSF10C, TRAIL/TNFSF10, TRANCE/RANK L/TNFSF11, 4-1BB Ligand/TNFSF9, TWEAK/TNFSF12, CD40 Ligand/TNFSF5, Fas Ligand/TNFSF6, RELT/TNFRSF19L, APRIL/TNFSF13, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, TRAIL R1/TNFRSF10A, TRAIL R4/TNFRSF10D, CD30 Ligand/TNFSF8, GITR Ligand/TNFSF18, TNFSF18, TACI/TNFRSF13B, NGF R/TNFRSF16, OX40 Ligand/TNFSF4, TRAIL R2/TNFRSF10B, TRAIL R3/TNFRSF10C, TWEAK R/TNFRSF12, BAFF/BLyS/TNFSF13, DR6/TNFRSF21, TNF-alpha/TNFSF1A, Pro-TNF-alpha/TNFSF1A, Lymphotoxin beta R/TNFRSF3, Lymphotoxin beta R (LTbR)/Fc Chimera, TNF RI/TNFRSF1A, TNF-beta/TNFSF1B, PGRP-S, TNF RI/TNFRSF1A, TNF RII/TNFRSF1B, EDA-A2, TNF-alpha/TNFSF1A, EDAR, XEDAR, TNF RI/TNFRSF1A.
- Of particular interest are human target proteins that are commercially available in purified form. Examples are: 4EBP1, 14-3-3 zeta, 53BP1, 2B4/SLAMF4, CCL21/6Ckine, 4-1BB/TNFRSF9, 8D6A, 4-1BB Ligand/TNFSF9, 8-oxo-dG, 4-Amino-1,8-naphthalimide, A2B5, Aminopeptidase LRAP/ERAP2, A33, Aminopeptidase N/ANPEP, Aag, Aminopeptidase P2/XPNPEP2, ABCG2, Aminopeptidase P1/XPNPEP1, ACE, Aminopeptidase PILS/ARTS1, ACE-2, Amnionless, Actin, Amphiregulin, beta-Actin, AMPK alpha 1/2, Activin A, AMPK alpha 1, Activin AB, AMPK alpha 2, Activin B, AMPK beta 1, Activin C, AMPK beta 2, Activin RIA/ALK-2, Androgen R/NR3C4, Activin RIB/ALK-4, Angiogenin, Activin RIIA, Angiopoietin-1, Activin RIIB, Angiopoietin-2, ADAM8, Angiopoietin-3, ADAM9, Angiopoietin-4, ADAM10, Angiopoietin-like 1, ADAM12, Angiopoietin-like 2, ADAM15, Angiopoietin-like 3, TACE/ADAM17, Angiopoietin-like 4, ADAM19, Angiopoietin-like 7/CDT6, ADAM33, Angiostatin, ADAMTS4, Annexin A1/Annexin I, ADAMTS5, Annexin A7, ADAMTS1, Annexin A10, ADAMTSL-1/Punctin, Annexin V, Adiponectin/Acrp30, ANP, AEBSF, AP Site, Aggrecan, APAF-1, Agrin, APC, AgRP, APE, AGTR-2, APJ, AIF, APLP-1, Akt, APLP-2, Akt1, Apolipoprotein AI, Akt2, Apolipoprotein B, Akt3, APP, Serum Albumin, APRIL/TNFSF13, ALCAM, ARC, ALK-1, Artemin, ALK-7, Arylsulfatase A/ARSA, Alkaline Phosphatase, ASAH2/N-acylsphingosine Amidohydrolase-2, alpha 2u-Globulin, ASC, alpha-1-Acid Glycoprotein, ASGR1, alpha-Fetoprotein, ASK1, ALS, ATM, Ameloblastin, ATRIP, AMICA/JAML, Aurora A, AMIGO, Aurora B, AMIGO2, Axin-1, AMIGO3, Ax1, Aminoacylase/ACY1, Azurocidin/CAP37/HBP, Aminopeptidase A/ENPEP, B4GALT1, BIM, B7-1/CD80, 6-Biotin-17-NAD, B7-2/CD86, BLAME/SLAMF8, B7-H1/PD-L1, CXCL13/BLC/BCA-1, B7-H2, BLIMP1, B7-H3, Blk, B7-H4, BMI-1, BACE-1, BMP-1/PCP, BACE-2, BMP-2, Bad, BMP-3, BAFF/TNFSF13B, BMP-3b/GDF-10, BAFF R/TNFRSF13C, BMP-4, Bag-1, BMP-5, BAK, BMP-6, BAMBI/NMA, BMP-7, BARD1, BMP-8, Bax, BMP-9, BCAM, BMP-10, Bcl-10, BMP-15/GDF-9B, Bcl-2, BMPR-IA/ALK-3, Bcl-2 related protein A1, BMPR-IB/ALK-6, Bcl-w, BMPR-II, Bcl-x, BNIP3L, Bcl-xL, BOC, BCMA/TNFRSF17, BOK, BDNF, BPDE, Benzamide, Brachyury, Common beta Chain, B-Raf, beta IG-H3, CXCL14/BRAK, Betacellulin, BRCA1, beta-Defensin 2, BRCA2, BID, BTLA, Biglycan, Bub-1, Bik-like Killer Protein, c-jun, CD90/Thy1, c-Rel, CD94, CCL6/C10, CD97, C1q R1/CD93, CD151, C1qTNF1, CD160, C1qTNF4, CD163, C1qTNF5, CD164, Complement Component C1r, CD200, Complement Component C1s, CD200 R1, Complement Component C2, CD229/SLAMF3, Complement Component C3a, CD23/Fc epsilon RII, Complement Component C3d, CD2F-10/SLAMF9, Complement Component C5a, CD5L, Cadherin-4/R-Cadherin, CD69, Cadherin-6, CDC2, Cadherin-8, CDC25A, Cadherin-11, CDC25B, Cadherin-12, CDCP1, Cadherin-13, CDO, Cadherin-17, CDX4, E-Cadherin, CEACAM-1/CD66a, N-Cadherin, CEACAM-6, P-Cadherin, Cerberus 1, VE-Cadherin, CFTR, Calbindin D, cGMP, Calcineurin A, Chem R23, Calcineurin B, Chemerin, Calreticulin-2, Chemokine Sampler Packs, CaM Kinase II, Chitinase 3-like 1, cAMP, Chitotriosidase/CHIT1, Cannabinoid R1, Chk1, Cannabinoid R2/CB2/CNR2, Chk2, CAR/NR1I3, CHL-1/L1CAM-2, Carbonic Anhydrase J, Choline Acetyltransferase/ChAT, Carbonic Anhydrase II, Chondrolectin, Carbonic Anhydrase III, Chordin, Carbonic Anhydrase IV, Chordin-Like 1, Carbonic Anhydrase VA, Chordin-Like 2, Carbonic Anhydrase VB, CINC-1, Carbonic Anhydrase VI, CINC-2, Carbonic Anhydrase VII, CINC-3, Carbonic Anhydrase VIII, Claspin, Carbonic Anhydrase IX, Claudin-6, Carbonic Anhydrase X, CLC, Carbonic Anhydrase XII, CLEC-1, Carbonic Anhydrase XIII, CLEC-2, Carbonic Anhydrase XIV, CLECSF13/CLEC4F, Carboxymethyl Lysine, CLECSF8, Carboxypeptidase A1/CPA1, CLF-1, Carboxypeptidase A2, CL-P1/COLEC12, Carboxypeptidase A4, Clusterin, Carboxypeptidase B1, Clusterin-like 1, Carboxypeptidase E/CPE, CMG-2, Carboxypeptidase XI, CMV UL146, Cardiotrophin-1, CMV UL147, Carnosine Dipeptidase 1, CNP, Caronte, CNTF, CART, CNTF R alpha, Caspase, Coagulation Factor II/Thrombin, Caspase-1, Coagulation Factor III/Tissue Factor, Caspase-2, Coagulation Factor VII, Caspase-3, Coagulation Factor X, Caspase-4, Coagulation Factor XI, Caspase-6, Coagulation Factor XIV/Protein C, Caspase-7, COCO, Caspase-8, Cohesin, Caspase-9, Collagen I, Caspase-10, Collagen II, Caspase-12, Collagen IV, Caspase-13, Common gamma Chain/IL-2 R gamma, Caspase Peptide Inhibitors, COMP/Thrombospondin-5, Catalase, Complement Component C1rLP, beta-Catenin, Complement Component C1qA, Cathepsin 1, Complement Component C1qC, Cathepsin 3, Complement Factor D, Cathepsin 6, Complement Factor I, Cathepsin A, Complement MASP3, Cathepsin B, Connexin 43, Cathepsin C/DPPI, Contactin-1, Cathepsin D, Contactin-2/TAG1, Cathepsin E, Contactin-4, Cathepsin F, Contactin-5, Cathepsin H, Corin, Cathepsin L, Cornulin, Cathepsin O, CORS26/C1qTNF, 3, Cathepsin S, Rat Cortical Stem Cells, Cathepsin V, Cortisol, Cathepsin X/Z/P, COUP-TF I/NR2F1, CBP, COUP-TF II/NR2F2, CCI, COX-1, CCK-A R, COX-2, CCL28, CRACC/SLAMF7, CCR1, C-Reactive Protein, CCR2, Creatine Kinase, Muscle/CKMM, CCR3, Creatinine, CCR4, CREB, CCR5, CREG, CCR6, CRELD1, CCR7, CRELD2, CCR8, CRHBP, CCR9, CRHR-1, CCR10, CRIM1, CD155/PVR, Cripto, CD2, CRISP-2, CD3, CRISP-3, CD4, Crossveinless-2, CD4+/45RA−, CRTAM, CD4+/45RO−, CRTH-2, CD4+/CD62L−/CD44, CRY1, CD4+/CD62L+/CD44, Cryptic, CD5, CSB/ERCC6, CD6, CCL27/CTACK, CD8, CTGF/CCN2, CD8+/45RA−, CTLA-4, CD8+/45RO−, Cubilin, CD9, CX3CR1, CD14, CXADR, CD27/TNFRSF7, CXCL16, CD27 Ligand/TNFSF7, CXCR3, CD28, CXCR4, CD30/TNFRSF8, CXCR5, CD30 Ligand/TNFSF8, CXCR6, CD31/PECAM-1, Cyclophilin A, CD34, Cyr61/CCN1, CD36/SR-B3, Cystatin A, CD38, Cystatin B, CD40/TNFRSF5, Cystatin C, CD40 Ligand/TNFSF5, Cystatin D, CD43, Cystatin E/M, CD44, Cystatin F, CD45, Cystatin H, CD46, Cystatin H2, CD47, Cystatin S, CD48/SLAMF2, Cystatin SA, CD55/DAF, Cystatin SN, CD58/LFA-3, Cytochrome c, CD59, Apocytochrome c, CD68, Holocytochrome c, CD72, Cytokeratin 8, CD74, Cytokeratin 14, CD83, Cytokeratin 19, CD84/SLAMF5, Cytonin, D6, DISP1, DAN, Dkk-1, DANCE, Dkk-2, DARPP-32, Dkk-3, DAX1/NR0B1, Dkk-4, DCC, DLEC, DCIR/CLEC4A, DLL1, DCAR, DLL4, DcR3/TNFRSF6B, d-Luciferin, DC-SIGN, DNA Ligase IV, DC-SIGNR/CD299, DNA Polymerase beta, DcTRAIL R1/TNFRSF23, DNAM-1, DcTRAIL R2/TNFRSF22, DNA-PKcs, DDR1, DNER, DDR2, Dopa Decarboxylase/DDC, DEC-205, DPCR-1, Decapentaplegic, DPP6, Decorin, DPPA4, Dectin-1/CLEC7A, DPPA5/ESG1, Dectin-2/CLEC6A, DPPII/QPP/DPP7, DEP-1/CD148, DPPIV/CD26, Desert Hedgehog, DR3/TNFRSF25, Desmin, DR6/TNFRSF21, Desmoglein-1, DSCAM, Desmoglein-2, DSCAM-L1, Desmoglein-3, DSPG3, Dishevelled-1, Dtk, Dishevelled-3, Dynamin, EAR2/NR2F6, EphA5, ECE-1, EphA6, ECE-2, EphA7, ECF-L/CHI3L3, EphA8, ECM-1, EphB1, Ecotin, EphB2, EDA, EphB3, EDA-A2, EphB4, EDAR, EphB6, EDG-1, Ephrin, EDG-5, Ephrin-A1, EDG-8, Ephrin-A2, eEF-2, Ephrin-A3, EGF, Ephrin-A4, EGF R, Ephrin-A5, EGR1, Ephrin-B, EG-VEGF/PK1, Ephrin-B1, eIF2 alpha, Ephrin-B2, eIF4E, Ephrin-B3, Elk-1, Epigen, EMAP-II, Epimorphin/Syntaxin 2, EMMPRIN/CD147, Epiregulin, CXCL5/ENA, EPR-1/Xa Receptor, Endocan, ErbB2, Endoglin/CD105, ErbB3, Endoglycan, ErbB4, Endonuclease III, ERCC1, Endonuclease IV, ERCC3, Endonuclease V, ERK1/ERK2, Endonuclease VIII, ERK1, Endorepellin/Perlecan, ERK2, Endostatin, ERK3, Endothelin-1, ERK5/BMK1, Engrailed-2, ERR alpha/NR3B1, EN-RAGE, ERR beta/NR3B2, Enteropeptidase/Enterokinase, ERR gamma/NR3B3, CCL11/Eotaxin, Erythropoietin, CCL24/Eotaxin-2, Erythropoietin R, CCL26/Eotaxin-3, ESAM, EpCAM/TROP-1, ER alpha/NR3A1, EPCR, ER beta/NR3A2, Eph, Exonuclease III, EphA1, Exostosin-like 2/EXTL2, EphA2, Exostosin-like 3/EXTL3, EphA3, FABP1, FGF-BP, FABP2, FGF R1-4, FABP3, FGF R1, FABP4, FGF R2, FABP5, FGF R3, FABP7, FGF R4, FABP9, FGF R5, Complement Factor B, Fgr, FADD, FHR5, FAM3A, Fibronectin, FAM3B, Ficolin-2, FAM3C, Ficolin-3, FAM3D, FITC, Fibroblast Activation Protein alpha/FAP, FKBP38, Fas/TNFRSF6, Flap, Fas Ligand/TNFSF6, FLIP, FATP1, FLRG, FATP4, FLRT1, FATP5, FLRT2, Fc gamma RI/CD64, FLRT3, Fc gamma RIIB/CD32b, Flt-3, Fc gamma RIIC/CD32c, Flt-3 Ligand, Fc gamma RIIA/CD32a, Follistatin, Fc gamma RIII/CD16, Follistatin-like 1, FcRH1/IRTA5, FosB/GOS3, FcRH2/IRTA4, FoxD3, FcRH4/IRTA1, FoxJ1, FcRH5/IRTA2, FoxP3, Fc Receptor-like 3/CD16-2, Fpg, FEN-1, FPR1, Fetuin A, FPRL1, Fetuin B, FPRL2, FGF acidic, CX3CL1/Fractalkine, FGF basic, Frizzled-1, FGF-3, Frizzled-2, FGF-4, Frizzled-3, FGF-5, Frizzled-4, FGF-6, Frizzled-5, FGF-8, Frizzled-6, FGF-9, Frizzled-7, FGF-10, Frizzled-8, FGF-11, Frizzled-9, FGF-12, Frk, FGF-13, sFRP-1, FGF-16, sFRP-2, FGF-17, sFRP-3, FGF-19, sFRP-4, FGF-20, Furin, FGF-21, FXR/NR1H4, FGF-22, Fyn, FGF-23, G9a/EHMT2, GFR alpha-3/GDNF R alpha-3, GABA-A-R alpha 1, GFR alpha-4/GDNF R alpha-4, GABA-A-R alpha 2, GITR/TNFRSF18, GABA-A-R alpha 4, GITR Ligand/TNFSF18, GABA-A-R alpha 5, GLI-1, GABA-A-R alpha 6, GLI-2, GABA-A-R beta 1, GLP/EHMT1, GABA-A-R beta 2, GLP-1 R, GABA-A-R beta 3, Glucagon, GABA-A-R gamma 2, Glucosamine (N-acetyl)-6-Sulfatase/GNS, GABA-B-R2, GluR1, GAD1/GAD67, GluR2/3, GAD2/GAD65, GluR2, GADD45 alpha, GluR3, GADD45 beta, Glut1, GADD45 gamma, Glut2, Galectin-1, Glut3, Galectin-2, Glut4, Galectin-3, Glut5, Galectin-3 BP, Glutaredoxin 1, Galectin-4, Glycine R, Galectin-7, Glycophorin A, Galectin-8, Glypican 2, Galectin-9, Glypican 3, GalNAc4S-6ST, Glypican 5, GAP-43, Glypican 6, GAPDH, GM-CSF, Gas1, GM-CSF R alpha, Gas6, GMF-beta, GASP-1/WFIKKNRP, gp130, GASP-2/WFIKKN; Glycogen Phosphorylase BB/GPBB, GATA-1, GPR15, GATA-2, GPR39, GATA-3, GPVI, GATA-4, GR/NR3C1, GATA-5, Gr-1/Ly-6G, GATA-6, Granulysin, GBL, Granzyme A, GCNF/NR6A1, Granzyme B, CXCL6/GCP-2, Granzyme D, G-CSF, Granzyme G, G-CSF R, Granzyme H, GDF-1, GRASP, GDF-3 GRB2, GDF-5, Gremlin, GDF-6, GRO, GDF-7, CXCL1/GRO alpha, GDF-8, CXCL2/GRO beta, GDF-9, CXCL3/GRO gamma, GDF-11, Growth Hormone, GDF-15, Growth Hormone R, GDNF, GRP75/HSPA9B, GFAP, GSK-3 alpha/beta, GFI-1, GSK-3 alpha, GFR alpha-1/GDNF R alpha-1, GSK-3 beta, GFR alpha-2/GDNF R alpha-2, EZF1T, H2AX, Histidine, H60, HM74A, HAI-1, HMGA2, HAI-2, HMGB1, HAI-2A, TCF-2/HNF-1 beta, HAI-2B, HNF-3 beta/FoxA2, HAND1, HNF-4 alpha/NR2A1, HAPLN1, HNF-4 gamma/NR2A2, Airway Trypsin-like Protease/HAT, HO-1/HMOX1/HSP32, HB-EGF, HO-2/HMOX2, CCL14a/HCC-1, HPRG, CCL14b/HCC-3, Hrk, CCL16/HCC-4, HRP-1, alpha HCG, HS6ST2, Hck, HSD-1, HCR/CRAM-A/B, HSD-2, HDGF, HSP10/EPF, Hemoglobin, HSP27, Hepassocin, HSP60, HES-1, HSP70, HES-4, HSP90, HGF, HTRA/Protease Do, HGF Activator, HTRA1/PRSS11, HGF R, HTRA2/Omi, HIF-1 alpha, HVEM/TNFRSF14, HIF-2 alpha, Hyaluronan, HIN-1/Secretoglobulin 3A1, 4-Hydroxynonenal, Hip, CCL1/I-309/TCA-3, IL-10, cIAP (pan), IL-10 R alpha, cIAP-1/HIAP-2, IL-10 R beta, cIAP-2/HIAP-1, IL-1, IBSP/Sialoprotein II, IL-11 R alpha, ICAM-1/CD54, IL-12, ICAM-2/CD102, IL-12/IL-23 p40, ICAM-3/CD50, IL-12 R beta 1, ICAM-5, IL-12 R beta 2, ICAT, IL-13, ICOS, IL-13 R alpha 1, Iduronate 2-Sulfatase/IDS, IL-13 R alpha 2, IFN, IL-15, IFN-alpha, IL-15 R alpha, IFN-alpha 1, IL-16, IFN-alpha 2, IL-17, IFN-alpha 4b, IL-17 R, IFN-alpha A, IL-17 RC, IFN-alpha B2, IL-17 RD, IFN-alpha C, IL-17B, IFN-alpha D, IL-17B R, IFN-alpha F, IL-17C, IFN-alpha G, IL-17D, IFN-alpha H2, IL-17E, IFN-alpha I, IL-17F, IFN-alpha J1, IL-18/IL-1F4, IFN-alpha K, IL-18 BPa, IFN-alpha WA, IL-18 BPc, IFN-alpha/beta R1, IL-18 BPd, IFN-alpha/beta R2, IL-18 R alpha/IL-1 R5, IFN-beta, IL-18 R beta/IL-1 R7, IFN-gamma, IL-19, IFN-gamma R1, IL-20, IFN-gamma R2, IL-20 R alpha, IFN-omega, IL-20 R beta, IgE, IL-21, IGFBP-1, IL-21 R, IGFBP-2, IL-22, IGFBP-3, IL-22 R, IGFBP-4, IL-22BP, IGFBP-5, IL-23, IGFBP-6, IL-23 R, IGFBP-L1, IL-24, IGFBP-rp1/IGFBP-7, IL-26/AK155, IGFBP-rP10, IL-27, IGF-I, IL-28A, IGF-I R, IL-28B, IGF-II, IL-29/IFN-lambda 1, IGF-II R, IL-31, IgG, IL-31 RA, IgM, IL-32 alpha, IGSF2, IL-33, IGSF4A/SynCAM, ILT2/CD85j, IGSF4B, ILT3/CD85k, IGSF8, ILT4/CD85d, IgY, ILT5/CD85a, IkB-beta, ILT6/CD85e, IKK alpha, Indian Hedgehog, IKK epsilon, INSRR, IKK gamma, Insulin, IL-1 alpha/IL-1F1, Insulin R/CD220, IL-1 beta/IL-1F2, Proinsulin, IL-1ra/IL-1F3, Insulysin/IDE, IL-1F5/FIL1 delta, Integrin alpha 2/CD49b, IL-1F6/FIL1 epsilon, Integrin alpha 3/CD49c, IL-1F7/FIL1 zeta, Integrin alpha 3 beta 1/VLA-3, IL-1F8/FIL1 eta, Integrin alpha 4/CD49d, IL-1F9/IL-1H1, Integrin alpha 5/CD49e, IL-1F10/IL-1HY2, Integrin alpha 5 beta 1, IL-1 RI, Integrin alpha 6/CD49f, IL-1 RII, Integrin alpha 7, IL-1 R3/IL-1 R AcP, Integrin alpha 9, IL-1 R4/ST2, Integrin alpha E/CD103, IL-1 R6/IL-1 R rp2, Integrin alpha L/CD11a, IL-1 R8, Integrin alpha L beta 2, IL-1 R9, Integrin alpha M/CD11b, IL-2, Integrin alpha M beta 2, IL-2 R alpha, Integrin alpha V/CD51, IL-2 R beta, Integrin alpha V beta 5, IL-3, Integrin alpha V beta 3, IL-3 R alpha, Integrin alpha V beta 6, IL-3 R beta, Integrin alpha X/CD11c, IL-4, Integrin beta 1/CD29, IL-4 R, Integrin beta 2/CD18, IL-5, Integrin beta 3/CD61, IL-5 R alpha, Integrin beta 5, IL-6, Integrin beta 6, IL-6 R, Integrin beta 7, IL-7, CXCL10/IP-10/CRG-2, IL-7 R alpha/CD127, IRAK1, CXCR1/IL-8 RA, IRAK4, CXCR2/IL-8 RB, IRS-1, CXCL8/IL-8, Islet-1, IL-9, CXCL11/I-TAC, IL-9 R, Jagged 1, JAM-4/IGSF5, Jagged 2, JNK, JAM-A, JNK1/JNK2, JAM-B/VE-JAM, JNK1, JAM-C, JNK2, Kininogen, Kallikrein 3/PSA, Kininostatin, Kallikrein 4, KIR/CD158, Kallikrein 5, KIR2DL1, Kallikrein 6/Neurosin, KIR2DL3, Kallikrein 7, KIR2DL4/CD158d, Kallikrein 8/Neuropsin, KIR2DS4, Kallikrein 9, KIR3DL1, Plasma Kallikrein/KLKB1, KIR3DL2, Kallikrein 10, Kirrel2, Kallikrein 11, KLF4, Kallikrein 12, KLF5, Kallikrein 13, KLF6, Kallikrein 14, Klotho, Kallikrein 15, Klotho beta, KC, KOR, Keap1, Kremen-1, Kell, Kremen-2, KGF/FGF-7, LAG-3, LINGO-2, LAIR1, Lipin 2, LAIR2, Lipocalin-1, Laminin alpha 4, Lipocalin-2/NGAL, Laminin gamma 1, 5-Lipoxygenase, Laminin I, LXR alpha/NR1H3, Laminin S, LXR beta/NR1H2, Laminin-1, Livin, Laminin-5, LIX, LAMP, LMIR1/CD300A, Langerin, LMIR2/CD300c, LAR, LMIR3/CD300LF, Latexin, LMIR5/CD300LB, Layilin, LMIR6/CD300LE, LBP, LMO2, LDL R, LOX-1/SR-E1, LECT2, LRH-1/NR5A2, LEDGF, LRIG1, Lefty, LRIG3, Lefty-1, LRP-1, Lefty-A, LRP-6, Legumain, LSECtin/CLEC4G, Leptin, Lumican, Leptin R, CXCL15/Lungkine, Leukotriene B4, XCL1/Lymphotactin, Leukotriene B4 R1, Lymphotoxin, LIF, Lymphotoxin beta/TNFSF3, LIF R alpha, Lymphotoxin beta R/TNFRSF3, LIGHT/TNFSF14, Lyn, Limitin, Lyp, LIMPII/SR-B2, Lysyl Oxidase Homolog 2, LIN-28, LYVE-1, LINGO-1, alpha 2-Macroglobulin, CXCL9/MIG, MAD2L1, Mimecan, MAdCAM-1, Mindin, MafB, Mineralocorticoid R/NR3C2, MafF, CCL3L1/MIP-1 alpha Isoform LD78 beta, MafG, CCL3/MIP-1 alpha, MafK, CCL4L1/LAG-1, MAG/Siglec-4a, CCL4/MIP-1 beta, MANF, CCL15/MIP-1 delta, MAP2, CCL9/10/MIP-1 gamma, MAPK, MIP-2, Marapsin/Pancreasin, CCL19/MIP-3 beta, MARCKS, CCL20/MIP-3 alpha, MARCO, MIP-I, Mash1, MIP-II, Matrilin-2, MIP-III, Matrilin-3, MIS/AMH, Matrilin-4, MIS RII, Matriptase/ST14, MIXL1, MBL, MKK3/MKK6, MBL-2, MKK3, Melanocortin 3R/MC3R, MKK4, MCAM/CD146, MKK6, MCK-2, MKK7, Mcl-1, MKP-3, MCP-6, MLH-1, CCL2/MCP-1, MLK4 alpha, MCP-11, MMP, CCL8/MCP-2, MMP-1, CCL7/MCP-3/MARC, MMP-2, CCL13/MCP-4, MMP-3, CCL12/MCP-5, MMP-7, M-CSF, MMP-8, M-CSF R, MMP-9, MCV-type 11, MMP-10, MD-1, MMP-11, MD-2; MMP-12, CCL22/MDC, MMP-13, MDL-1/CLEC5A, MMP-14, MDM2, MMP-15, MEA-1, MMP-16/MT3-MMP, MEK1/MEK2, MMP-24/MT5-MMP, MEK1, MMP-25/MT6-MMP, MEK2, MMP-26, Melusin, MMR, MEPE, MOG, Meprin alpha, CCL23/MPIF-1, Meprin beta, M-Ras/R-Ras3, Mer, Mre11, Mesothelin, MRP1 Meteorin, MSK1/MSK2, Methionine Aminopeptidase 1, MSK1, Methionine Aminopeptidase, MSK2, Methionine Aminopeptidase 2, MSP, MFG-E8, MSP R/Ron, MFRP, Mug, MgcRacGAP, MULT-1, MGL2, Musashi-1, MGMT, Musashi-2, MIA, MuSK, MICA, MutY DNA Glycosylase, MICB, MyD88, MICL/CLEC12A, Myeloperoxidase, beta 2 Microglobulin, Myocardin, Midkine, Myocilin, MIF, Myoglobin, NAIP NGFI-B gamma/NR4A3, Nanog, NgR2/NgRH1, CXCL7/NAP-2, NgR3/NgRH2, Nbs1, Nidogen-1/Entactin, NCAM-1/CD56, Nidogen-2, NCAM-L1, Nitric Oxide, Nectin-1, Nitrotyrosine, Nectin-2/CD112, NKG2A, Nectin-3, NKG2C, Nectin-4, NKG2D, Neogenin, NKp30, Neprilysin/CD10, NKp44, Neprilysin-2/MMEL1/MMEL2, NKp46/NCR1, Nestin, NKp80/KLRF1, NETO2, NKX2.5, Netrin-1, NMDA R, NR1 Subunit, Netrin-2, NMDA R, NR2A Subunit, Netrin-4, NMDA R, NR2B Subunit, Netrin-G1a, NMDA R, NR2C Subunit, Netrin-G2a, N-Me-6,7-diOH-TIQ, Neuregulin-1/NRG1, Nodal, Neuregulin-3/NRG3, Noggin, Neuritin, Nogo Receptor, NeuroD1, Nogo-A, Neurofascin, NOMO, Neurogenin-1, Nope, Neurogenin-2, Norrin, Neurogenin-3, eNOS, Neurolysin, iNOS, Neurophysin II, nNOS, Neuropilin-1, Notch-1, Neuropilin-2, Notch-2, Neuropoietin, Notch-3, Neurotrimin, Notch-4, Neurturin, NOV/CCN3, NFAM1, NRAGE, NF-H, NrCAM, NFkB1, NRL, NFkB2, NT-3, NF-L, NT-4, NF-M, NTB-A/SLAMF6, NG2/MCSP, NTH1, NGF R/TNFRSF16, Nucleostemin, beta-NGF, Nurr-1/NR4A2, NGFI-B alpha/NR4A1, OAS2, Orexin B, OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIL/CLEC2d, Oncostatin M/OSM, OCILRP2/CLEC21, OSM R beta, Oct-3/4, Osteoactivin/GPNMB, OGG1, Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin, Olig2, Osteopontin, Olig3, Osteoprotegerin/TNFRSF11B, Oligodendrocyte Marker O1, Otx2, Oligodendrocyte Marker O4, OV-6, OMgp, OX40/TNFRSF4, Opticin, OX40 Ligand/TNFSF4, Orexin A, OAS2, Orexin B, OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIL/CLEC2d, Oncostatin M/OSM, OCILRP2/CLEC2i, OSM R beta, Oct-3/4, Osteoactivin/GPNMB, OGG1, Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin, Olig2, Osteopontin, Olig3, Osteoprotegerin/TNFRSF11B, Oligodendrocyte Marker O1, Otx2, Oligodendrocyte Marker O4, OV-6, OMgp, OX40/TNFRSF4, Opticin, OX40 Ligand/TNFSF4, Orexin A, RACK1, Ret, Rad1, REV-ERB alpha/NR1D1, Rad17, REV-ERB beta/NR1D2, Rad51, Rex-1, Rae-1, RGM-A, Rae-1 alpha, RGM-B, Rae-1 beta, RGM-C, Rae-1 delta, Rheb, Rae-1 epsilon, Ribosomal Protein S6, Rae-1 gamma, RIP1, Raf-1, ROBO1, RAGE, ROBO2, Ra1A/Ra1B, ROBO3, Ra1A, ROBO4, Ra1B, ROR/NR1F1-3 (pan), RANK/TNFRSF11A, ROR alpha/NR1F1, CCL5/RANTES, ROR gamma/NR1F3, Rap1A/B, RTK-like Orphan Receptor 1/ROR1, RAR alpha/NR1B1, RTK-like Orphan Receptor 2/ROR2, RAR beta/NR1B2, RP105, RAR gamma/NR1B3, RPA2, Ras, RSK (pan), RBP4, RSK1/RSK2, RECK, RSK1, Reg 2/PAP, RSK2, Reg I, RSK3, Reg II, RSK4, Reg III, R-Spondin 1, Reg IIIa, R-Spondin 2, Reg IV, R-Spondin 3, Relaxin-1, RUNX1/CBFA2, Relaxin-2, RUNX2/CBFA1, Relaxin-3, RUNX3/CBFA3, RELM alpha, RXR alpha/NR2B1, RELM beta, RXR beta/NR2B2, RELT/TNFRSF19L, RXR gamma/NR2B3, Resistin, S100A10, SLITRK5, S100A8, SLP1, S100A9, SMAC/Diablo, S100B, Smad1, S100P, Smad2, SALL1, Smad3, delta-Sarcoglycan, Smad4, Sca-1/Ly6, Smad5, SCD-1, Smad7, SCF, Smad8, SCF R/c-kit, SMC1, SCGF, alpha-Smooth Muscle Actin, SCL/Tal1, SMUG1, SCP3/SYCP3, Snail, CXCL12/SDF-1, Sodium Calcium Exchanger 1, SDNSF/MCFD2, Soggy-1, alpha-Secretase, Sonic Hedgehog, gamma-Secretase, S or CSI, beta-Secretase, S or CS3, E-Selectin, Sortilin, L-Selectin, SOST, P-Selectin, SOX1, Semaphorin 3A, SOX2, Semaphorin 3C, SOX3, Semaphorin 3E, SOX7, Semaphorin 3F, SOX9, Semaphorin 6A, SOX10, Semaphorin 6B, SOX17, Semaphorin 6C, SOX21 Semaphorin 6D, SPARC, Semaphorin 7A, SPARC-like 1, Separase, SP-D, Serine/Threonine Phosphatase Substrate I, Spinesin, Serpin A1, F-Spondin, Serpin A3, SR-AI/MSR, Serpin A4/Kallistatin, Src, Serpin A5/Protein C Inhibitor, SREC-1/SR-F1, Serpin A8/Angiotensinogen, SREC-II, Serpin B5, SSEA-1, Serpin C1/Antithrombin-III, SSEA-3, Serpin D1/Heparin Cofactor II, SSEA-4, Serpin E1/PAI-1, ST7/LRP12, Serpin E2, Stabilin-1, Serpin F1, Stabilin-2, Serpin F2, Stanniocalcin 1, Serpin G1/C1 Inhibitor, Stanniocalcin 2, Serpin 12, STAT1, Serum Amyloid A1, STAT2, SF-1/NR5A1, STAT3, SGK, STAT4, SHBG, STAT5a/b, SHIP, STAT5a, SHP/NROB2, STAT5b, SHP-1, STAT6, SHP-2, VE-Statin, SIGIRR, Stella/Dppa3, Siglec-2/CD22, STRO-1, Siglec-3/CD33, Substance P, Siglec-5, Sulfamidase/SGSH, Siglec-6, Sulfatase Modifying Factor 1/SUMF1, Siglec-7, Sulfatase Modifying Factor 2/SUMF2, Siglec-9, SUMO1, Siglec-10, SUMO2/3/4, Siglec-11, SUMO3, Siglec-F, Superoxide Dismutase, SIGNR1/CD209, Superoxide Dismutase-1/Cu—Zn SOD, SIGNR4, Superoxide Dismutase-2/Mn-SOD, SIRP beta 1, Superoxide Dismutase-3/EC-SOD, SKI, Survivin, SLAM/CD150, Synapsin I, Sleeping Beauty Transposase, Syndecan-1/CD138, Slit3, Syndecan-2, SLITRK1, Syndecan-3, SLITRK2, Syndecan-4, SLITRK4, TACI/TNFRSF13B, TMEFF1/Tomoregulin-1, TAO2, TMEFF2, TAPP1, TNF-alpha/TNFSF1A, CCL17/TARC, TNF-beta/TNFSF1B, Tau, TNF RI/TNFRSF1A, TC21/R-Ras2, TNF RII/TNFRSF1B, TCAM-1, TOR, TCCR/WSX-1, TP-1, TC-PTP, TP63/TP73L, TDG, TR, CCL25/TECK, TR alpha/NR1A1, Tenascin C, TR beta 1/NR1A2, Tenascin R, TR2/NR2C1, TER-119, TR4NR2C2, TERT, TRA-1-85, Testican 1/SPOCK1, TRADD, Testican 2/SPOCK2, TRAF-1, Testican 3/SPOCK3, TRAF-2, TFPI, TRAF-3, TFPI-2, TRAF-4, TGF-alpha, TRAF-6, TGF-beta, TRAIL/TNFSF10, TGF-beta 1, TRAIL R1/TNFRSF10A, LAP (TGF-beta 1), TRAIL R2/TNFRSF10B, Latent TGF-beta 1, TRAIL R3/TNFRSF10C, TGF-beta 1.2, TRAIL R4/TNFRSF10D, TGF-beta 2, TRANCE/TNFSF11, TGF-beta 3, TfR (Transferrin R), TGF-beta 5, Apo-Transferrin, Latent TGF-beta bp1, Holo-Transferrin, Latent TGF-beta bp2, Trappin-2/Elafin, Latent TGF-beta bp4, TREM-1, TGF-beta R/ALK-5, TREM-2, TGF-beta RII, TREM-3, TGF-beta RIIb, TREML1/TLT-1, TGF-beta RIII, TRF-1, Thermolysin, TRF-2, Thioredoxin-1, TRH-degrading Ectoenzyme/TRHDE, Thioredoxin-2, TRIM5, Thioredoxin-80, Tripeptidyl-Peptidase I, Thioredoxin-like 5/TRP14, TrkA, THOP1, TrkB, Thrombomodulin/CD141, TrkC, Thrombopoietin, TROP-2, Thrombopoietin R, Troponin I Peptide 3, Thrombospondin-1, Troponin T, Thrombospondin-2, TROY/TNFRSF19, Thrombospondin-4, Trypsin 1, Thymopoietin, Trypsin 2/PRSS2, Thymus Chemokine-1, Trypsin 3/PRSS3, Tie-1, Tryptase-5/Prss32, Tie-2, Tryptase alpha/TPS1, TIM-1/KIM-1/HAVCR, Tryptasebeta-1/MCPT-7, TIM-2, Tryptasebeta-2/TPSB2, TIM-3, Tryptase epsilon/BSSP-4, TIM-4, Tryptase gamma-1/TPSG1, TIM-5, Tryptophan Hydroxylase, TIM-6, TSC22, TIMP-1, TSG, TIMP-2, TSG-6, TIMP-3, TSK, TIMP-4, TSLP, TL1A/TNFSF15, TSLP R, TLR1, TSP50, TLR2, beta-III Tubulin, TLR3, TWEAK/TNFSF12, TLR4, TWEAK R/TNFRSF12, TLR5, Tyk2, TLR6, Phospho-Tyrosine, TLR9, Tyrosine Hydroxylase, TLX/NR2E1, Tyrosine Phosphatase Substrate I, Ubiquitin, UNC5H3, Ugi, UNC5H4, UGRP1, UNG, ULBP-1, uPA, ULBP-2, uPAR, ULBP-3, URB, UNC5H1, UVDE, UNC5H2, Vanilloid R1, VEGF R, VASA, VEGF R1/Flt-1, Vasohibin, VEGF R2/KDR/Flk-1, Vasorin, VEGF R3/Flt-4, Vasostatin, Versican, Vav-1, VG5Q, VCAM-1, VHR, VDR/NRIII, Vimentin, VEGF, Vitronectin, VEGF-B, VLDLR, VEGF-C, vWF-A2, VEGF-D, Synuclein-alpha, Ku70, WASP, Wnt-7b, WIF-1, Wnt-8a WISP-1/CCN4, Wnt-8b, WNK1, Wnt-9a, Wnt-1, Wnt-9b, Wnt-3a, Wnt-10a, Wnt-4, Wnt-10b, Wnt-5a, Wnt-11, Wnt-5b, wnvNS3, Wnt7a, XCR1, XPE/DDBI, XEDAR, XPE/DDB2, Xg, XPF, XIAP, XPG, XPA, XPV, XPD, XRCC1, Yes, YY1, EphA4.
- Numerous human ion channels are targets of particular interest. Non-limiting examples include 5-hydroxytryptamine 3 receptor B subunit, 5-hydroxytryptamine 3 receptor precursor, 5-hydroxytryptamine receptor 3 subunit C, AAD14 protein, Acetylcholine receptor protein, alpha subunit precursor, Acetylcholine receptor protein, beta subunit precursor, Acetylcholine receptor protein, delta subunit precursor, Acetylcholine receptor protein, epsilon subunit precursor, Acetylcholine receptor protein, gamma subunit precursor, Acid sensing ion channel 3 splice variant b, Acid sensing ion channel 3 splice variant c, Acid sensing ion channel 4, ADP-ribose pyrophosphatase, mitochondrial precursor, Alpha1A-voltage-dependent calcium channel, Amiloride-sensitive cation channel 1, neuronal, Amiloride-sensitive cation channel 2, neuronal Amiloride-sensitive cation channel 4, isoform 2, Amiloride-sensitive sodium channel, Amiloride-sensitive sodium channel alpha-subunit, Amiloride-sensitive sodium channel beta-subunit, Amiloride-sensitive sodium channel delta-subunit, Amiloride-sensitive sodium channel gamma-subunit, Annexin A7, Apical-like protein, ATP-sensitive inward rectifier potassium channel 1, ATP-sensitive inward rectifier potassium channel 10, ATP-sensitive inward rectifier potassium channel 11, ATP-sensitive inward rectifier potassium channel 14, ATP-sensitive inward rectifier potassium channel 15, ATP-sensitive inward rectifier potassium channel 8, Calcium channel alpha12.2 subunit, Calcium channel alpha12.2 subunit, Calcium channel alpha1E subunit, delta19 delta40 delta46 splice variant, Calcium-activated potassium channel alpha subunit 1, Calcium-activated potassium channel beta subunit 1, Calcium-activated potassium channel beta subunit 2, Calcium-activated potassium channel beta subunit 3, Calcium-dependent chloride channel-1, Cation channel TRPM4B, CDNA FLJ90453 fis, clone NT2RP3001542, highly similar to Potassium channel tetramerisation domain containing 6, CDNA FLJ90663 fis, clone PLACE1005031, highly similar to Chloride intracellular channel protein 5, CGMP-gated cation channel beta subunit, Chloride channel protein, Chloride channel protein 2, Chloride channel protein 3, Chloride channel protein 4, Chloride channel protein 5, Chloride channel protein 6, Chloride channel protein ClC-Ka, Chloride channel protein ClC-Kb, Chloride channel protein, skeletal muscle, Chloride intracellular channel 6, Chloride intracellular channel protein 3, Chloride intracellular channel protein 4, Chloride intracellular channel protein 5, CHRNA3 protein, Clcn3e protein, CLCNKB protein, CNGA4 protein, Cullin-5, Cyclic GMP gated potassium channel, Cyclic-nucleotide-gated cation channel 4, Cyclic-nucleotide-gated cation channel alpha 3, Cyclic-nucleotide-gated cation channel beta 3, Cyclic-nucleotide-gated olfactory channel, Cystic fibrosis transmembrane conductance regulator, Cytochrome B-245 heavy chain, Dihydropyridine-sensitive L-type, calcium channel alpha-2/delta subunits precursor, FXYD domain-containing ion transport regulator 3 precursor, FXYD domain-containing ion transport regulator 5 precursor, FXYD domain-containing ion transport regulator 6 precursor, FXYD domain-containing ion transport regulator 7, FXYD domain-containing ion transport regulator 8 precursor, G protein-activated inward rectifier potassium channel 1, G protein-activated inward rectifier potassium channel 2, G protein-activated inward rectifier potassium channel 3, G protein-activated inward rectifier potassium channel 4, Gamma-aminobutyric-acid receptor alpha-1 subunit precursor, Gamma-aminobutyric-acid receptor alpha-2 subunit precursor, Gamma-aminobutyric-acid receptor alpha-3 subunit precursor, Gamma-aminobutyric-acid receptor alpha-4 subunit precursor, Gamma-aminobutyric-acid receptor alpha-5 subunit precursor, Gamma-aminobutyric-acid receptor alpha-6 subunit precursor, Gamma-aminobutyric-acid receptor beta-1 subunit precursor, Gamma-aminobutyric-acid receptor beta-2 subunit precursor, Gamma-aminobutyric-acid receptor beta-3 subunit precursor, Gamma-aminobutyric-acid receptor delta subunit precursor, Gamma-aminobutyric-acid receptor epsilon subunit precursor, Gamma-aminobutyric-acid receptor gamma-1 subunit precursor, Gamma-aminobutyric-acid receptor gamma-3 subunit precursor, Gamma-aminobutyric-acid receptor pi subunit precursor, Gamma-aminobutyric-acid receptor rho-1 subunit precursor, Gamma-aminobutyric-acid receptor rho-2 subunit precursor, Gamma-aminobutyric-acid receptor theta subunit precursor, GluR6 kainate receptor, Glutamate receptor 1 precursor, Glutamate receptor 2 precursor, Glutamate receptor 3 precursor, Glutamate receptor 4 precursor, Glutamate receptor 7, Glutamate receptor B, Glutamate receptor delta-1 subunit precursor, Glutamate receptor, ionotropic kainate 1 precursor, Glutamate receptor, ionotropic kainate 2 precursor, Glutamate receptor, ionotropic kainate 3 precursor, Glutamate receptor, ionotropic kainate 4 precursor, Glutamate receptor, ionotropic kainate 5 precursor, Glutamate [NMDA] receptor subunit 3A precursor, Glutamate [NMDA] receptor subunit 3B precursor, Glutamate [NMDA] receptor subunit epsilon 1 precursor, Glutamate [NMDA] receptor subunit epsilon 2 precursor, Glutamate [NMDA] receptor subunit epsilon 4 precursor, Glutamate [NMDA] receptor subunit zeta 1 precursor, Glycine receptor alpha-1 chain precursor, Glycine receptor alpha-2 chain precursor, Glycine receptor alpha-3 chain precursor, Glycine receptor beta chain precursor, H/ACA ribonucleoprotein complex subunit 1, High affinity immunoglobulin epsilon receptor beta-subunit, Hypothetical protein DKFZp31310334, Hypothetical protein DKFZp761M1724, Hypothetical protein FLJ12242, Hypothetical protein FLJ14389, Hypothetical protein FLJ14798, Hypothetical protein FLJ14995, Hypothetical protein FLJ16180, Hypothetical protein FLJ16802, Hypothetical protein FLJ32069, Hypothetical protein FLJ37401, Hypothetical protein FLJ38750, Hypothetical protein FLJ40162, Hypothetical protein FLJ41415, Hypothetical protein FLJ90576, Hypothetical protein FLJ90590, Hypothetical protein FLJ90622, Hypothetical protein KCTD15, Hypothetical protein MGC15619, Inositol 1,4,5-trisphosphate receptor type 1, Inositol 1,4,5-trisphosphate receptor type 2, Inositol 1,4,5-trisphosphate receptor type 3, Intermediate conductance calcium-activated potassium channel protein 4, Inward rectifier potassium channel 13, Inward rectifier potassium channel 16, Inward rectifier potassium channel 4, Inward rectifying K(+) channel negative regulator Kir2.2v, Kainate receptor subunit KA2a, KCNH5 protein, KCTD17 protein, KCTD2 protein, Keratinocytes associated transmembrane protein 1, Kv channel-interacting protein 4, Melastatin 1, Membrane protein MLC1, MGC15619 protein, Mucolipin-1, Mucolipin-2, Mucolipin-3, Multidrug resistance-associated protein 4, N-methyl-D-aspartate receptor 2C subunit precursor, NADPH oxidase homolog 1, Nav1.5, Neuronal acetylcholine receptor protein, alpha-10 subunit precursor, Neuronal acetylcholine receptor protein, alpha-2 subunit precursor, Neuronal acetylcholine receptor protein, alpha-3 subunit precursor, Neuronal acetylcholine receptor protein, alpha-4 subunit precursor, Neuronal acetylcholine receptor protein, alpha-5 subunit precursor, Neuronal acetylcholine receptor protein, alpha-6 subunit precursor, Neuronal acetylcholine receptor protein, alpha-7 subunit precursor, Neuronal acetylcholine receptor protein, alpha-9 subunit precursor, Neuronal acetylcholine receptor protein, beta-2 subunit precursor, Neuronal acetylcholine receptor protein, beta-3 subunit precursor, Neuronal acetylcholine receptor protein, beta-4 subunit precursor, Neuronal voltage-dependent calcium channel alpha 2D subunit, P2X purinoceptor 1, P2X purinoceptor 2, P2X purinoceptor 3, P2X purinoceptor 4, P2X purinoceptor 5, P2X purinoceptor 6, P2X purinoceptor 7, Pancreatic potassium channel TALK-1b, Pancreatic potassium channel TALK-1c, Pancreatic potassium channel TALK-1d, Phospholemman precursor, Plasmolipin, Polycystic kidney disease 2 related protein, Polycystic kidney disease 2-like 1 protein, Polycystic kidney disease 2-like 2 protein, Polycystic kidney disease and receptor for egg jelly related protein precursor, Polycystin-2, Potassium channel regulator, Potassium channel subfamily K member 1, Potassium channel subfamily K member 10, Potassium channel subfamily K member 12, Potassium channel subfamily K member 13, Potassium channel subfamily K member 15, Potassium channel subfamily K member 16, Potassium channel subfamily K member 17, Potassium channel subfamily K member 2, Potassium channel subfamily K member 3, Potassium channel subfamily K member 4, Potassium channel subfamily K member 5, Potassium channel subfamily K member 6, Potassium channel subfamily K member 7, Potassium channel subfamily K member 9, Potassium channel tetramerisation domain containing 3, Potassium channel tetramerisation domain containing protein 12, Potassium channel tetramerisation domain containing protein 14, Potassium channel tetramerisation domain containing protein 2, Potassium channel tetramerisation domain containing protein 4, Potassium channel tetramerisation domain containing protein 5, Potassium channel tetramerization domain containing 10, Potassium channel tetramerization domain containing protein 13, Potassium channel tetramerization domain-containing 1, Potassium voltage-gated channel subfamily A member 1, Potassium voltage-gated channel subfamily A member 2, Potassium voltage-gated channel subfamily A member 4, Potassium voltage-gated channel subfamily A member 5, Potassium voltage-gated channel subfamily A member 6, Potassium voltage-gated channel subfamily B member 1, Potassium voltage-gated channel subfamily B member 2, Potassium voltage-gated channel subfamily C member 1, Potassium voltage-gated channel subfamily C member 3, Potassium voltage-gated channel subfamily C member 4, Potassium voltage-gated channel subfamily D member 1, Potassium voltage-gated channel subfamily D member 2, Potassium voltage-gated channel subfamily D member 3, Potassium voltage-gated channel subfamily E member 1, Potassium voltage-gated channel subfamily E member 2, Potassium voltage-gated channel subfamily E member 3, Potassium voltage-gated channel subfamily E member 4, Potassium voltage-gated channel subfamily F member 1, Potassium voltage-gated channel subfamily E member 1, Potassium voltage-gated channel subfamily G member 2, Potassium voltage-gated channel subfamily G member 3, Potassium voltage-gated channel subfamily G member 4, Potassium voltage-gated channel subfamily H member 1, Potassium voltage-gated channel subfamily H member 2, Potassium voltage-gated channel subfamily H member 3, Potassium voltage-gated channel subfamily H member 4, Potassium voltage-gated channel subfamily H member 5, Potassium voltage-gated channel subfamily H member 6, Potassium voltage-gated channel subfamily H member 7, Potassium voltage-gated channel subfamily H member 8, Potassium voltage-gated channel subfamily KQT member 1, Potassium voltage-gated channel subfamily KQT member 2, Potassium voltage-gated channel subfamily KQT member 3, Potassium voltage-gated channel subfamily KQT member 4, Potassium voltage-gated channel subfamily KQT member 5, Potassium voltage-gated channel subfamily S member 1, Potassium voltage-gated channel subfamily S member 2, Potassium voltage-gated channel subfamily S member 3, Potassium voltage-gated channel subfamily V member 2, Potassium voltage-gated channel, subfamily H, member 7, isoform 2, Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1, Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2, Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 3, Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, Probable mitochondrial import receptor subunit TOM40 homolog, Purinergic receptor P2X5, isoform A, Putative 4 repeat voltage-gated ion channel, Putative chloride channel protein 7, Putative GluR6 kainate receptor, Putative ion channel protein CATSPER2 variant 1, Putative ion channel protein CATSPER2 variant 2, Putative ion channel protein CATSPER2 variant 3, Putative regulator of potassium channels protein variant 1, Putative tyrosine-protein phosphatase TPTE, Ryanodine receptor 1, Ryanodine receptor 2, Ryanodine receptor 3, SH3 KBP1 binding protein 1, Short transient receptor potential channel 1, Short transient receptor potential channel 4, Short transient receptor potential channel 5, Short transient receptor potential channel 6, Short transient receptor potential channel 7, Small conductance calcium-activated potassium channel protein 1, Small conductance calcium-activated potassium channel protein 2, isoform b, Small conductance calcium-activated potassium channel protein 3, isoform b, Small-conductance calcium-activated potassium channel SK2, Small-conductance calcium-activated potassium channel SK3, Sodium channel, Sodium channel beta-1 subunit precursor, Sodium channel protein type II alpha subunit, Sodium channel protein type III alpha subunit, Sodium channel protein type IV alpha subunit, Sodium channel protein type IX alpha subunit, Sodium channel protein type V alpha subunit, Sodium channel protein type VII alpha subunit, Sodium channel protein type VIII alpha subunit, Sodium channel protein type X alpha subunit, Sodium channel protein type XI alpha subunit, Sodium- and chloride-activated ATP-sensitive potassium channel, Sodium/potassium-transporting ATPase gamma chain, Sperm-associated cation channel 1, Sperm-associated cation channel 2, isoform 4, Syntaxin-1B1, Transient receptor potential cation channel subfamily A member 1, Transient receptor potential cation channel subfamily M member 2, Transient receptor potential cation channel subfamily M member 3, Transient receptor potential cation channel subfamily M member 6, Transient receptor potential cation channel subfamily M member 7, Transient receptor potential cation channel subfamily V member 1, Transient receptor potential cation channel subfamily V member 2, Transient receptor potential cation channel subfamily V member 3, Transient receptor potential cation channel subfamily V member 4, Transient receptor potential cation channel subfamily V member 5, Transient receptor potential cation channel subfamily V member 6, Transient receptor potential channel 4 epsilon splice variant, Transient receptor potential channel 4 zeta splice variant, Transient receptor potential channel 7 gamma splice variant, Tumor necrosis factor, alpha-induced protein 1, endothelial, Two-pore calcium channel protein 2, VDAC4 protein, Voltage gated potassium channel Kv3.2b, Voltage gated sodium channel beta 1B subunit, Voltage-dependent anion channel, Voltage-dependent anion channel 2, Voltage-dependent anion-selective channel protein 1, Voltage-dependent anion-selective channel protein 2, Voltage-dependent anion-selective channel protein 3, Voltage-dependent calcium channel gamma-1 subunit, Voltage-dependent calcium channel gamma-2 subunit, Voltage-dependent calcium channel gamma-3 subunit, Voltage-dependent calcium channel gamma-4 subunit, Voltage-dependent calcium channel gamma-5 subunit, Voltage-dependent calcium channel gamma-6 subunit, Voltage-dependent calcium channel gamma-7 subunit, Voltage-dependent calcium channel gamma-8 subunit, Voltage-dependent L-type calcium channel alpha-1C subunit, Voltage-dependent L-type calcium channel alpha-1D subunit, Voltage-dependent L-type calcium channel alpha-1S subunit, Voltage-dependent L-type calcium channel beta-1 subunit, Voltage-dependent L-type calcium channel beta-2 subunit, Voltage-dependent L-type calcium channel beta-3 subunit, Voltage-dependent L-type calcium channel beta-4 subunit, Voltage-dependent N-type calcium channel alpha-1B subunit, Voltage-dependent P/Q-type calcium channel alpha-1A subunit, Voltage-dependent R-type calcium channel alpha-1E subunit, Voltage-dependent T-type calcium channel alpha-1G subunit, Voltage-dependent T-type calcium channel alpha-1H subunit, Voltage-dependent T-type calcium channel alpha-1I subunit, Voltage-gated L-type calcium channel alpha-1 subunit, Voltage-gated potassium channel beta-1 subunit, Voltage-gated potassium channel beta-2 subunit, Voltage-gated potassium channel beta-3 subunit, Voltage-gated potassium channel KCNA7.
- Exemplary GPCRs include but are not limited to Class A Rhodopsin like receptors such as Musc. acetylcholine
Vertebrate type 1, Musc. acetylcholineVertebrate type 2, Musc. acetylcholineVertebrate type 3, Musc. acetylcholine Vertebrate type 4; Adrenoceptors (Alpha Adrenoceptors type 1, Alpha Adrenoceptors type 2, Beta Adrenoceptors type 1, Beta Adrenoceptors type 2, Beta Adrenoceptors type 3, Dopamine Vertebrate type 1, Dopamine Vertebrate type 2, Dopamine Vertebrate type 3, Dopamine Vertebrate type 4, Histamine type 1, Histamine type 2, Histamine type 3, Histamine type 4, Serotonin type 1, Serotonin type 2, Serotonin type 3, Serotonin type 4, Serotonin type 5, Serotonin type 6, Serotonin type 7, Serotonin type 8, other Serotonin types, Trace amine, Angiotensin type 1, Angiotensin type 2, Bombesin, Bradykinin, C5a anaphylatoxin, Finet-leu-phe, APJ like, Interleukin-8 type A, Interleukin-8 type B, Interleukin-8 type others, C—C Chemokine type 1 through type 11 and other types, C—X—C Chemokine (types 2 through 6 and others), C—X3-C Chemokine, Cholecystokinin CCK, CCK type A, CCK type B, CCK others, Endothelin, Melanocortin (Melanocyte stimulating hormone, Adrenocorticotropic hormone, Melanocortin hormone), Duffy antigen, Prolactin-releasing peptide (GPR10), Neuropeptide Y (type 1 through 7), Neuropeptide Y, Neuropeptide Y other, Neurotensin, Opioid (type D, K, M, X), Somatostatin (type 1 through 5), Tachykinin (Substance P (NK1), Substance K (NK2), Neuromedin K (NK3), Tachykinin like 1, Tachykinin like 2, Vasopressin/vasotocin (type 1 through 2), Vasotocin, Oxytocin/mesotocin, Conopressin, Galanin like, Proteinase-activated like, Orexin & neuropeptides FF, QRFP, Chemokine receptor-like, Neuromedin U like (Neuromedin U, PRXamide), hormone protein (Follicle stimulating hormone, Lutropin-choriogonadotropic hormone, Thyrotropin, Gonadotropin type I, Gonadotropin type II), (Rhod)opsin, Rhodopsin Vertebrate (types 1-5), Rhodopsin Vertebrate type 5, Rhodopsin Arthropod, Rhodopsin Arthropod type 1, Rhodopsin Arthropod type 2, Rhodopsin Arthropod type 3, Rhodopsin Mollusc, Rhodopsin, Olfactory (Olfactory II fam 1 through 13), Prostaglandin (prostaglandin E2 subtype EP1, Prostaglandin E2/D2 subtype EP2, prostaglandin E2 subtype EP3, Prostaglandin E2 subtype EP4, Prostaglandin F2-alpha, Prostacyclin, Thromboxane, Adenosine type 1 through 3, Purinoceptors, Purinoceptor P2RY1-4,6,11 GPR91, Purinoceptor P2RY5,8,9,10 GPR35,92,174, Purinoceptor P2RY12-14 GPR87 (UDP-Glucose), Cannabinoid, Platelet activating factor, Gonadotropin-releasing hormone, Gonadotropin-releasing hormone type I, Gonadotropin-releasing hormone type II, Adipokinetic hormone like, Corazonin, Thyrotropin-releasing hormone & Secretagogue, Thyrotropin-releasing hormone, Growth hormone secretagogue, Growth hormone secretagogue like, Ecdysis-triggering hormone (ETHR), Melatonin, Lysosphingolipid & LPA (EDG), Sphingosine 1-phosphate Edg-1, Lysophosphatidic acid Edg-2, Sphingosine 1-phosphate Edg-3, Lysophosphatidic acid Edg-4, Sphingosine 1-phosphate Edg-5, Sphingosine 1-phosphate Edg-6, Lysophosphatidic acid Edg-7, Sphingosine 1-phosphate Edg-8, Edg Other Leukotriene B4 receptor, Leukotriene B4 receptor BLT1, Leukotriene B4 receptor BLT2, Class A Orphan/other, Putative neurotransmitters, SREB, Mas proto-oncogene & Mas-related (MRGs), GPR45 like, Cysteinyl leukotriene, G-protein coupled bile acid receptor, Free fatty acid receptor (GP40, GP41, GP43), Class B Secretin like, Calcitonin, Corticotropin releasing factor, Gastric inhibitory peptide, Glucagon, Growth hormone-releasing hormone, Parathyroid hormone, PACAP, Secretin, Vasoactive intestinal polypeptide, Latrophilin, Latrophilin type 1, Latrophilin type 2, Latrophilin type 3, ETL receptors, Brain-specific angiogenesis inhibitor (BAI), Methuselah-like proteins (MTH), Cadherin EGF LAG (CELSR), Very large G-protein coupled receptor, Class C Metabotropic glutamate/pheromone, Metabotropic glutamate group I through III, Calcium-sensing like, Extracellular calcium-sensing, Pheromone, calcium-sensing like other, Putative pheromone receptors, GABA-B, GABA-B subtype 1, GABA-B subtype 2, GABA-B like, Orphan GPRC5, Orphan GPCR6, Bride of sevenless proteins (BOSS), Taste receptors (TIR), Class D Fungal pheromone, Fungal pheromone A-Factor like (STE2,STE3), Fungal pheromone B like (BAR,BBR,RCB,PRA), Class E cAMP receptors, Ocular albinism proteins, Frizzled/Smoothened family, frizzled Group A (Fz 1&2&4&5&7-9), frizzled Group B (Fz 3 & 6), frizzled Group C (other), Vomeronasal receptors, Nematode chemoreceptors, Insect odorant receptors, and Class Z Archaeal/bacterial/fungal opsins. - The subject MURPs can be designed to target any cellular proteins including but not limited to cell surface protein, secreted protein, cytosolic protein, and nuclear protein. A target of particular interest is an ion channel.
- Ion channels constitute a superfamily of proteins, including the family of potassium channels (K-channels), the family of sodium channels (Na-channels), the family of calcium channels (Ca-channels), the family of Chlorine channels (Cl-channels) and the family of acetylcholine channels. Each of these families contains subfamilies and each subfamily typically contains specific channels derived from single genes. For example, the K-channel family contains subfamilies of voltage-gated K-channels called Kv1.x and Kv3.x. The subfamily Kv1.x contains the channels Kv1.1, Kv1.2 and Kv1.3, which correspond to the products of single genes and are thus called ‘species’. The classification applies to the Na-, Ca-, Cl- and other families of channels as well.
- Ion channels can also be classified according to the mechanisms by which the channels are operated. Specifically, the main types of ion channel proteins are characterized by the method employed to open or close the channel protein to either permit or prevent specific ions from permeating the channel protein and crossing a lipid bilayer cellular membrane. One important type of channel protein is the voltage-gated channel protein, which is opened or closed (gated) in response to changes in electrical potential across the cell membrane. The voltage-gated sodium channel 1.6 (Nav1.6) is of particular interest as a therapeutic target. Another type of ion channel protein is the mechanically gated channel, for which a mechanical stress on the protein opens or closes the channel. Still another type is called a ligand-gated channel, which opens or closes depending on whether a particular ligand is bound to the protein. The ligand can be either an extracellular moiety, such as a neurotransmitter, or an intracellular moiety, such as an ion or nucleotide.
- Ion channels generally permit passive flow of ions down an electrochemical gradient, whereas ion pumps use ATP to transport against a gradient. Coupled transporters, both antiporters and symporters, allow movement of one ion species against its gradient, powered by the downhill movement of another ion species.
- One of the most common types of channel proteins, found in the membrane of almost all animal cells, permits the specific permeation of potassium ions across a cell membrane. In particular, potassium ions permeate rapidly across cell membranes through K+ channel proteins (up to 10−8 ions per second). Moreover, potassium channel proteins have the ability to distinguish among potassium ions, and other small alkali metal ions, such as Li+ or Na+ with great fidelity. In particular, potassium ions are at least ten thousand times more permanent than sodium ions. Potassium channel proteins typically comprise four (usually identical) subunits, so their cell surface targets are present as tetramers, allowing tetravalent binding of MURPs. One type of subunit contains six long hydrophobic segments (which can be membrane-spanning), while the other types contains two hydrophobic segments.
- Another significant family of channels is calcium channel. Calcium channels are generally classified according to their electrophysiological properties as Low-voltage-activated (LVA) or High-voltage-activated (HVA) channels. HVA channels comprises at least three groups of channels, known as L-, N- and P/Q-type channels. These channels have been distinguished one from another electrophysiologically as well as bio-chemically on the basis of their pharmacology and ligand binding properties. For instance, dihydropyridines, diphenyl-alkylamines and piperidines bind to the α1 subunit of the L-type calcium channel and block a proportion of HVA calcium currents in neuronal tissue, which are termed L-type calcium currents. N-type calcium channels are sensitive to omega conopeptides, but are relatively insensitive to dihydropyridine compounds, such as nimodipine and nifedipine. P/Q-type channels, on the other hand, are insensitive to dihydropyridines, but are sensitive to the funnel web spider toxin Aga IIIA. R-type calcium channels, like L-, N-, P- and Q-type channels, are activated by large membrane depolarizations, and are thus classified as high voltage-activated (HVA) channels. R-type channels are generally insensitive to dihydropyridines and omega conopeptides, but, like P/Q, L and N channels, are sensitive to the funnel web spider toxin AgaIVA. Immunocytochemical staining studies indicate that these channels are located throughout the brain, particularly in deep midline structures (caudate-putamen, thalamus, hypothalamus, amygdala, cerebellum) and in the nuclei of the ventral midbrain and brainstem. Neuronal voltage-sensitive calcium channels typically consists of a central α1. subunit, an α2/δ subunit, a β subunit and a 95 kD subunit.
- Additional non-limiting examples include Kir (an inwardly rectified potassium channel), Kv (a voltage-gated potassium channel), Nav (a voltage-gated sodium channel), Cav (a voltage-gated calcium channel), CNG (cyclic nucleotide-gated channel), HCN (hyperpolarization-activated channel), TRP (a transient receptor potential channel), ClC (a chloride channel), CFTR (a cystic fibrosis transmembrane conductance regulator, a chloride channel), IP3R (a inositol trisphosphate receptor), RYR (a ryanodine receptor). Other channel types are 2-pore channels, glutamate-receptors (AMPA, NMDA, KA), M2, Connexins and Cys-loop receptors.
- A common layout for ion channel proteins, such as Kv1.2, Kv3.1, Shaker; TRPC1 and TRPC5 is to have six membrane-spanning segments, arranged as follows:
-
- N-terminus---S1---E1---S2---X1---S3---E2---S4---X2---S5---E3---S6---C-terminus
- Wherein S1-6 are membrane-spanning sequences, E1-3 are extracellular surface loops and X1-2 are intracellular surface loops. The E3 loop is generally the longest of the three extracellular loops and is hydrophilic so it is a good target for drugs and MURPs to bind. The pore-forming part of most channels is a multimeric (e.g. tetrameric or rarely pentameric) complex of membrane-spanning alpha-helices. There is generally a pore loop, which is a region of the protein that loops back into the membrane to form the selectivity filter that determines which ion species can permeate. Such channels are called ‘pore-loop’ channels.
- The ion channels are valuable targets for drug design because they are involved in a broad range of physiological processes. In human, there exist approximately over three hundreds of ion channel proteins, many of which have been implicated in genetic diseases. For example, abbrebrant expression or function of ion channels has been shown to cause a wide arrange of diseases including cardiac, neuronal, muscular, respiratory metabolic diseases. This section focuses on ion channels, but the same concepts and approaches are equally applicable to all membrane proteins, including 7TMs, 1TMs, G-proteins and G-Protein Coupled receptors (GPCRs), etc. Some of the ion channels are GPCRs.
- Ion channels typically form large macromolecular complexes that include tightly bound accessory protein subunits and combinatorial use of such subunits contributes to the diversity of ion channels. These accessory proteins can also be the binding targets of the subject MURPs, microproteins and toxins.
- The subject MURPs can be designed to bind any of the channels known in the art and to those specifically exemplified herein. MURPs exhibiting a desired ion channel binding capability (encompassing specificity and avidity) can be selected by any recombinant and biochemical (e.g. expression and display) techniques known in the art. For instance, MURPs can be displayed by a genetic package including but not limited to phages and spores, and be subjected to panning against intact cell membranes, or preferably intact cells such as whole mammalian cells. To remove the phage that bind to the other, non-target cell surface molecules, the standard approach was to perform subtraction panning against similar cell lines that had a low or non-detectable level of the target receptor. However, Popkov et al. (J. Immunol. Methods 291:137-151 (2004)) showed that related cell types are not ideal for subtraction because they generally have a reduced but still significant level of the target on their surface, which reduces the number of desired phage clones. This problem occurs even when panning on cells that have been transfected with the gene encoding the target, followed by negative selection/subtraction on the same cell-line which was not transfected, especially when the native target gene was not knocked out. Instead, Popkov et al. showed that the negative selection or subtraction panning works much better if performed with an excess of the same cells that are used for normal panning (positive selection), except that the target has now been blocked with a high-affinity, target-specific inhibitor, such as a small molecule, peptide or an antibody to the target, which makes the active site unavailable. This process is called “negative selection with epitope-masked cells”, which is particularly useful in selecting the subject MURPs with a desired ion-channel binding capability.
- In a separate embodiment, the present invention provides microproteins, and particularly microproteins exhibiting binding capability towards at least one family of ion channels. The present invention also provides a genetic package displaying such microproteins. Non-limiting ion-channel examples to which the subject microproteins bind are sodium, potassium, calcium, acetylcholine, and chlorine channels. Of particular interest are those microproteins and the genetic packages displaying such microproteins, which exhibit binding capability towards native targets. Native targets are generally natural molecules or fragments, derivatives thereof that the microprotein is known to bind, typically including those known binding targets that have been reported in the literature.
- The subject invention also provides a genetic package displaying an ion-channel-binding microprotein which has been modified. The modified microprotein may (a) binds to a different family of channel as compared to the corresponding unmodified microprotein; (b) binds to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein; (c) binds to a different species of the same subfamily of channel as compared to the corresponding unmodified microprotein; (d) the microprotein binds to a different site on the same channel as compared to the corresponding unmodified microprotein; and/or (e) binds to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein.
-
FIGS. 22 and 46 show how microprotein domains or toxins that each bind at different sites of the same ion channel can be combined into a single protein. The two binding sites that these two microproteins bind to can be on two channels from different families, two channels from the same family but a different subfamily, two channels from the same subfamily but a different species (gene product), or two different binding sites on the same channel (species) or they can (simultaneously or not) bind the same binding site on the same channel (species) since the channels are multimeric. The binding modules and domains that bind to sites on the channels can be microprotein domains (natural or non-natural, 2- to 8-disulfide containing), one-disulfide peptides, or linear peptides. These modules can be selected independently and combined, or one can be selected from a library to bind in the presence of one fixed, active binding module. In the latter case, the display library would display multiple modules of which one would contain a library of variants. A typical goal is to select a dimer from this library that has a higher affinity than the active monomer that was the starting point. - In another embodiment, the present invention provides a protein comprising a plurality of ion-channel binding domains, wherein individual domains are microprotein domains that have been modified such that (a) the microprotein domains bind to a different family of channel as compared to the corresponding unmodified microprotein domains; (b) the microprotein domains bind to a different subfamily of the same channel family as compared to the corresponding unmodified microprotein domains; (c) the microprotein domains bind to a different species of the same subfamily as compared to the corresponding unmodified microprotein domains; (d) the microprotein domains bind to a different site on the same channel as compared to the corresponding unmodified microprotein domains; (e) the microprotein domains bind to the same site of the same channel but yield a different biological effect as compared to the corresponding unmodified microprotein domains; and/or (f) the microprotein domains bind to the same site of the same channel and yield the same biological effect as compared to the corresponding unmodified microprotein domains. Where desired, the microprotein domains may comprise natural or non-natural sequences. The individual domains can be linked together via a heterologous linker. The individual microprotein domains can bind to the same or different channel family, same or different channel subfamily, same or different species of the same subfamily, same or different site on the same channel.
- The subject microproteins can be a toxin. Preferably, the toxin retains in part or in whole its toxicity spectrum. In particular, venomous animals, such as snakes, encounter a range of prey and intruder species and the venom toxins differ in activity for the different receptors of the different species. The venom consists of a large number of related and unrelated toxins, with each toxin having a “spectrum of activity”, which can be defined as all of the receptors from all of the species on which that toxin has measurable activity. All of the targets in the ‘spectrum of activity’ are considered “native targets” and this includes any human targets that the toxin is active against. The native target(s) of a microprotein or toxin include all of the targets that the toxin is reported to inhibit in the literature. The higher the affinity or activity on a target, the more likely that target is the natural, native target, but it is not uncommon for toxins to act on multiple targets within the same species. Native target(s) can be human or non-human receptors that the toxin is active against.
- For the toxin to retain the ability to bind to cells after fusion to the display vector, it may be desirable to test both the N-terminus and C-terminus for fusion and to test a variety of fusion sites (i.e., 0, 1, 2, 3, 4, 5, 6 amino acids before the first cysteine or after the last cysteine of the toxin domain, if the toxin domain is a cysteine-containing domain) using a synthetic DNA library approach, preferably encoding a library of glycine-rich linkers, which form the smallest amino acid chain, are uncharged and are most likely to be compatible with binding of the toxin to the target. Since the N-terminal amino group and the C-terminal carboxyl groups may be involved in target binding, the library should contain a lysine or a arginine to mimic the positively charged amino group (or fusions to the N-terminus of the toxin) and a glutamate or an aspartate to mimic the negatively charged carboxyl group (for fusions to the C-terminus of the toxin).
- The inhibitor(s) that are used to block the target during negative selection can be small molecules, peptides or proteins, and natural or non-natural. In addition to simple subtraction, the choice of the mixture of inhibitors is a valuable tool to control the specificity of the ion channel inhibitors that are being designed. Because there are over three hundreds ion channels in total, with partially overlapping specificities and sequence similarities, and multiple modulatory sites per channel, each having a different effect, the specificity requirement can be complex.
- When modifying the activity of a toxin, or when combining two different toxins into a single protein, the two toxins can bind the same channel at the same site and have the same physiologic effect, or the two toxins can bind the same channel at the same site and have a different physiologic effect, or the two toxins can bind to the same channel at a different site, or the two toxins can bind to different channels that belong to the same subfamily (i.e. Kv1.3 and Kv1.2; meaning product of a different gene or ‘species’), or the two toxins can bind to different channels that belong to the same family (i.e. both are K-channels), or the two toxins can bind to channels that belong to different families (i.e. K-channels versus Na-channels).
- Ion channels typically have many transmembrane segments (24 for sodium channels) and thus offer a number of different, non-competing and non-overlapping binding sites for modulators to alter the activity of the channel in different ways. One approach is to create binders for one site on the same ion channel from existing binders for a different site, even if these sites are unrelated. To achieve this, the existing toxin can be used as a targeting agent for a library of 1-, 2-, 3-, or 4-disulfide proteins that is separated from the targeting toxin by a flexible linker of 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40 or 50 amino acids. It is useful if the affinity of the targeting agent is not too high, so that the affinity of the new library can have a significant contribution to the overall affinity. Another approach is to create new modulators for channels from existing modulators for other channels that are related in sequence or in structure. The conotoxin family, for example, contains sequence-related and structure-related modulators for Ca-, K, Na-channels and nicotinic acetylcholine receptors. It appears feasible to convert a K-channel modulator into a Na-channel modulator using a library of conotoxin-derivatives, or vice versa. For example, Kappa-conotoxins inhibit K-channels, Mu-conotoxins and Delta-conotoxins inhibit Na-channels, Omega-conotoxins inhibit Ca-channels and Alpha-conotoxins inhibit acetylcholine receptors.
- The proximity of different binding sites, each with a different effect on channel activity, from the same ion channel makes it attractive to link the inhibitors using flexible linkers, creating a single inhibitor with two domains, each binding at a different site. Or a single protein with two domains that bind at different copies of the same site, yielding a bivalent, high affinity interaction (avidity). This approach has not been taken by natural toxins, presumably because they must act fast and thus stay small in order to have maximal tissue penetration, but for pharmaceuticals the speed of action is less important, making this is an attractive approach.
- One can thus create combinatorial libraries of dimeric, trimeric, tetrameric or multimeric toxins/modulators, each native or modified, and directly screen these libraries at the protein level or pan these libraries using genetic packages for improved affinity (avidity, if binding occurs simultaneously at multiple sites) and then characterize the specificity and activity of such multimeric clones by protein expression and purification followed by cell-based activity assays, including patch-clamp assays. The individual modules can be panned and selected separately, in isolation of each other, or they can be designed in each other's presence, such that the new domain is added to a display system as a library that also contain a fixed, active copy that serves as a targeting element for the library and only clones that are significantly better than the fixed, active monomer are selected and characterized.
-
FIGS. 46 and 47 show some of the monomeric derivatives that can be made from native (natural) toxins, and some of the multimers that can be made to bind at multiple different binding sites of the target. The linkers are shown as glycine-rich rPEG, but the linkers could be any sequence and could also be optimized using molecular libraries followed by panning. One can create libraries inside the active, native toxin itself, using a variety of mutagenesis strategies as describes above, or one can expand the existing area of contact with the target by creating libraries on the N-terminal or C-terminal side of the active toxin, hoping to create additional contacts with the target. Such libraries can be based on existing toxins with known activity for that site, or they can be or naive 1-, 2-, 3-, 4-disulfide libraries based on unrelated microprotein scaffolds. These additional contact elements can be added on one or both sides of the active domains, and can be directly adjacent to the existing modulatory domain or they can be separated from it by flexible linkers. The initial multimer or the final, improved multimer can be a homomultimer or a heteromultimer, based on sequence similarity of the domains or based on target specificity of the domains of the multimer. Thus, the monomers that comprise the multimer may bind to the same target sites but have the same or different sequences. With 10-100 different native toxins that are known to bind to each family of channels, and with 2, 3, 4, 5 or 6 domains per clone, display libraries with a huge combinatorial diversity can be created even if one only uses native toxin sequences. Low level synthetic mutagenesis based on amino acid similarity or on phylogenetic substitution rates within the family can be used to create high quality libraries of mutants, of which a very high fraction is expected to retain function, with a high probability of enhanced function in some of the properties of interest. - The binding capability of the subject MURPs, microproteins, or toxins to a given ion channel can be measured in terms of Hill Coefficient. Hill Coefficient indicates the stoichiometry of the binding interaction. A Hill coefficient of 2 indicates that 2 inhibitors bind to each channel. One can also assess the allosteric modulation, which is modulation of activity at one site caused by binding at a distant site.
- The biological activity or effect of an ion channel and the ability of the subject MURPs, microproteins or toxins to regulate an ion channel activity can be assessed using a variety of in vitro and in vivo assays. For instance, methods are available in the art for measuring voltage, measuring current, measuring membrane potential, measuring ion flux, e.g., potassium or rubidium, measuring ion concentration, measuring gating, measuring second messengers and transcription levels, and using e.g., voltage-sensitive dyes, radioactive tracers, and patch-clamp electrophysiology. In particular such assays can be used to test for microproteins and toxins that can inhibit or activate an ion channel of interest.
- Specifically, potential channel inhibitors or activators can be tested in comparison to a suitable control to examine the extent of modulation. Control samples can also be samples untreated with the candidate activators or inhibitors. Inhibition is present when a given ion channel activity value relative to the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or even less. IC50 is a commonly used unit (the concentration of inhibitor that reduces the ion channel's activity by 50%) for determining the inhibitory effect. Similar for IC90. Activation of channels is achieved when the select a given ion channel activity value relative to the control is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more.
- Changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing the channel of interest. For instance, one method is to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents are conveniently determined using the standard methodology (see, e.g., Hamil et al., Pflugers. Archiv. 391:85 (1981). Other known assays include: radiolabeled rubidium flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70 (1994)).
- The effects of the candidate MURPs, microproteins, or toxins upon the function of a channel of interest can be measured by changes in the electrical currents or ionic flux or by the consequences of changes in currents and flux. The downstream effect of the candidate proteins on ion flux can be varied. Accordingly, any suitable physiological change can be used to assess the influence of a candidate protein on the test channels. The effects of candidate protein can be measured by a toxin binding assay. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release (e.g., dopamine), hormone release (e.g., insulin), transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), cell volume changes (e.g., in red blood cells), immunoresponses (e.g., T cell activation), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca2+.
- Other key biological activities of ion channels are ion selectivity and gating. Selectivity is the ability of some channels to discriminate between ion species, allowing some to pass through the pore while excluding others. Gating is the transition between open and closed states. They can be assessed by any of the methods known in the art or disclosed herein
- Yet another biological property that the subject MURP, microprotein, or toxin can be selected for is the frequency of opening and closing of the target channels, called Gating Frequency. Gating Frequency is influenced by voltage (in voltage gated channels, which are opened or closed by changes in membrane voltage) and ligand-binding. The transition rate between open and closed states is typically <10 microseconds but can be increased or decreased by other molecules. The flux rate (current) through the pore when it is open is on the order of 10e7 ions per second for ion channels and much less for coupled exchangers. Following opening, some voltage-gated channels enter an inactivated, non-conducting state in which they are refractory to depolarization.
- The human genome data base was searched for sequences that are rich in glycine. Three sequences were identified as suitable donor sequences as shown in Table X.
-
TABLE X Donor sequences for GRS design A. Amino Accession Sequences acid Protein NP_009060 GGGSGGGSGSGGGG 486-499 zinc finger protein Q9Y2X9 GSGSGGGGSGG 19-31 zinc finger protein CAG38801 SGGGGSGGGSGSG 7-19 MAP2K4 - Based on the sequences in Table X we designed a glycine rich sequence that contains multiple repeats of the peptide A with sequence GGGSGSGGGGS. Peptide A can be oligomerized to form structures with the formula (GGGSGSGGGGS)n where n is between 2 and 40.
FIG. 5 shows that all possible 9mer subsequences in oligomers of peptide A are contained in at least one of the proteins listed in table 3. Thus oligomers of peptide A do not contain human T cell epitopes. Inspection ofFIG. 5 reveals that GRS based on oligomers of peptide A can begin and end at any of the positions of peptide A. - Glycine rich sequences were designed based on sequence GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG, which represents amino acids 146-182 of the
human class 4 POU domain with accession number NP—006228.FIG. 6 illustrates that oligomers of peptide B with sequence GGGGGPGGGGP can be utilized as GRS. All 9mer subsequences that are contained in peptides with the sequence (GGGGGPGGGGP)n are also contained in the sequence of the POU domain. Thus, such oligomeric sequences do not contain T cell epitopes. - Glycine rich sequences can be designed based on the subsequence GAGGEGGGGEGGGPGG that is part of the ribosomal protein S6 kinase (accession number BAD92170). For instance, oligomers of peptide C with the sequence GGGGE will form sequences where most 9mer subsequences will be contained in the sequence of ribosomal protein S6 kinase. Thus, oligomeric GRS of the general structure (GGGGE)n bear a very low risk of containing T cell epitopes.
- A data base of human proteins was searched for subsequences that are rich in glycine residues. These subsequences contained at least 50% glycine. Only the following non-glycine residues were allowed to occur in the GRS: ADEHKPRST. 70 subsequences were identified that had a minimum length of 20 amino acids. These subsequences are listed in appendix A. They can be utilized to construct GRS with low immunogenic potential in humans.
- The following example describes the construction of a codon optimized gene encoding a URP sequence with 288 amino acids and the sequence (GSGGEG)48. First we constructed a stuffer vector pCW0051 as illustrated in
FIG. 40 . The sequence of the expression cassette in pCW0051 is shown inFIG. 42 . The stuffer vector was based on a pET vector and includes a T7 promoter. The vector encodes a Flag sequence followed by a stuffer sequence that is flanked by BsaI, BbsI, and KpnI sites. The BsaI and BbsI sites were inserted such that they generate compatible overhangs after digestion as illustrated inFIG. 42 . The stuffer sequence was followed by a His6 tag and the gene of green fluorescent protein (GFP). The stuffer sequence contains stop codons and thus E. coli cells carrying the stuffer plasmid pCW0051 formed non-fluorescent colonies. The stuffer vector pCW0051 was digested with BsaI and KpnI. A codon library encoding URP sequences of 36 amino acid length was constructed as shown inFIG. 41 . The URP sequence was designated rPEG_J36 and had the amino acid sequence (GSGGEG)6. The insert was obtained by annealing synthetic oligonucleotide pairs encoding the amino acid sequence GSGGEGGSGGEG as well as a pair of oligonucleotides that encode an adaptor to the KpnI site. The following oligonucleotides were used: pr_LCW0057 for: AGGTAGTGGWGGWGARGGWGGWTCYGGWGGAGAAGG, pr_LCW0057rev: - ACCTCCTTCTCCWCCRGAWCCWCCYTCWCCWCCACT,
pr —3 KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC,pr —3 KpnIstopperRev: CCTCGAGTGAAGACGA. The annealed oligonucleotide pairs were ligated, which resulted in a mixture of products with varying length that represents the varying number of rPEG_J12 repeats. The product corresponding to the length of rPEG_J36 was isolated from the mixture by agarose gel electrophoresis and ligated into the BsaI/KpnI digested stuffer vector pCW0051. Most of the clones in the resulting library designated LCW0057 showed green fluorescence after induction which shows that the sequence of rPEG_J36 had been ligated in frame with the GFP gene. The process of screening and iterative multimerization of rPEG_J36 sequences is illustrated inFIG. 14 . We screened 288 isolates from library LCW0057 for high level of fluorescence. 48 isolates with strong fluorescence were analyzed by PCR to verify the length of the rPEG_J segment and 16 clones were identified that had the expected length of rPEG_J36. This process resulted in a collection of 16 isolates of rPEG_J36, which show high expression and which differ in their codon usage. The isolates were pooled and dimerized using a process outlined inFIG. 40 . A plasmid mixture was digested with BsaI/NcoI and a fragment comprising the rPEG_J36 sequence and a part of GFP was isolated. The same plasmid mixture was also digested with BbsI/NcoI and the vector fragment comprising rPEG_J36, most of the plasmid vector, and the remainder of the GFP gene was isolated. Both fragments were mixed, ligated, and transformed into BL21 and isolates were screened for fluorescence. This process of dimerization was repeated two more rounds as outlined inFIG. 14 . During each round, we doubled the length of the rPEG_J gene and ultimately obtained a collection of genes that encode rPEG_J288. The amino acid and nucleotide sequence of rPEG_J288 is shown inFIG. 15 . It can be seen that the rPEG_J288 module contains segments of rPEG_J36 that differ in their nucleotide sequence despite of having identical amino acid sequence. Thus we minimized internal homology in the gene and as a result we reduced the risk of spontaneous recombination. We cultured E. coli BL21 harboring plasmids encoding rPEG_J288 for at least 20 doublings and no spontaneous recombination was observed. - A library of genes encoding a 288 amino acid URP termed rPEG_H288 was constructed using the same procedure that was used to construct rPEG_J288. rPEG_H288 has the amino acid sequence (GSGGEGGSGGSG)24. The flow chart of the construction process in shown in
FIG. 14 . The complete amino acid sequence as well as the nucleotide sequence of one isolate of rPEG_H288 as given inFIG. 16 . - A fusion protein containing the an N-terminal Flag tag and the URP sequence rPEG_J288 fused to the N-terminus of green fluorescent protein was incubated in 50% mouse serum at 37 C for 3 days. Samples were withdrawn at various time points and analyzed by SDS PAGE followed by detection using Western analysis. An antibody against the N-terminal flag tag was used for Western detection. Results are shown in
FIG. 28 , which indicate that a URP sequence of 288 amino acids can be completely stable in serum for at least three days. - Existence of antibodies against URP would be an indication of a potential immunogenic response to this glycine rich sequence. To test for the presence of existing antibodies in serum, an URP-GFP fusion was subjected to an ELISA by immobilizing URP-GFP on a support and subsequently incubating with 30% serum. The presence of antibodies bound to URP-GFP were detected using an anti-IgG-horse radish peroxidase antibody and substrate. The data are shown in
FIG. 29 . The data show, that the fusion protein can be detected by antibodies against GFP or Flag but not by murine serum. This indicates that murine serum does not contain antibodies that contain the URP sequence. - We purified a protein with the architecture Flag-rPEG_J288-H6-GFP. The protein was expressed in E. coli BL21 in SB medium. Cultures were induced with 0.5 mM IPTG overnight at 18 C. Cells were harvested by centrifugation. The pellet was re-suspended in TBS buffer containing benzonase and a commercial protease inhibitor cocktail. The suspension was heated for 10 min at 75 C in a water bath to lyze the cells. Insoluble material was removed by centrifugation. The supernatant was purified using immobilized metal ion specificity (IMAC) followed by a column with immobilized anti-Flag antibody.
FIG. 43 shows PAGE analysis of the purification process. The process yielded protein with at least 90% purity. - A gene encoding human interferon alpha was designed using codon optimization for E. coli expression. The synthetic gene was fused with a gene encoding rPEG_J288. A His6 tag was placed at the N-terminus to facilitate detection and purification, of the fusion protein. The amino acid sequence of the fusion protein is given in
FIG. 44 . - A gene encoding human G-CSF was designed using codon optimization for E. coli expression. The synthetic gene was fused with a gene encoding rPEG_J288. A His6 tag was placed at the N-terminus to facilitate detection and purification of the fusion protein. The amino acid sequence of the fusion protein is given in
FIG. 44 . - A gene encoding human growth hormone was designed using codon optimization for E. coli expression. The synthetic gene was fused with a gene encoding rPEG_J288. A His6 tag was placed at the N-terminus to facilitate detection and purification of the fusion protein. The amino acid sequence of the fusion protein is given in
FIG. 44 . - The fusion proteins between rPEG_J288 and two human proteins, interferon-alpha and human growth hormone were cloned into a T7 expression vector and transformed into E. coli BL21. The cells were grown at 37 C to an optical density of 0.5 OD. Subsequently, the cells were cultured at 18 C for 30 min. Then 0.5 mM IPTG was added and the cultures were incubated in a shaking incubator at 18 C overnight. Cells were harvested by centrifugation and soluble protein was released using BugBuster (Novagen). Both, insoluble and soluble protein fractions were separated by SDS-PAGE and the fusion proteins were detected by Western using and antibody against the N-terminal His6 tag for detection.
FIG. 45 shows the Western analysis of the two fusion proteins as well as rPEG_J288-GFP as control. All fusion proteins were expressed and the majority of the protein was in the soluble fraction. This is evidence of the high solubility of rPEG_J288 because most attempts at expression of the interferon-alpha and human growth hormone in the cytosol of E. coli, that have been reported in the literature, resulted in the formation of insoluble inclusion bodies.FIG. 45 shows that the majority of fusion proteins are expressed as full length proteins, i.e. no fragments that would suggest incomplete synthesis or partial protein degradation were detected. - Libraries of cysteine-constrained peptides were constructed as published [Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51]. These libraries were panned against human VEGF and two binding modules were indentified consisting of amino acid sequences FTCTNHWCPS or FQCTRHWCPI. Oligonucleotides encoding the amino acid sequence FTCTNHWCPS were ligated to a nucleotide sequence encoding the URP sequence rPEG_A36 with the sequence (GGS)12. Subsequently, the fusion sequence was dimerized using restriction enzymes and ligation steps to construct a molecule that contains 4 copies of the VEGF binding module separated by rPEG_A36 fused to GFP. The VEGF binding affinity of fusion proteins containing between zero and four VEGF-binding units were compared in
FIG. 30 . A fusion protein containing only rPEG_A36 fused to GFP shows no affinity for VEGF. Adding increasing numbers of VEGF binding modules increases affinity of the resulting fusion proteins. - Random peptide libraries were generated according to Scholle, et al. [Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51]. The naïve peptide libraries displayed cysteine-constrained peptides with cysteines spaced by 4 to 10 random residues. The library design is illustrated in the table:
-
TABLE X Naive 1SS libraries: LNG0001 XXXCXXCXXX X3CX2CX3 NNS NNS NNS TGC NNS NNS TGT NNS NNS NNS LNG0002 XXCXXXCXXX X2CX3CX3 NNS NNS TGC NNS NNS NNS TGT NNS NNS NNS LNG0003 XXCXXXXCXX X2CX4CX2 NNS NNS TGC NNS NNS NNS NNS TGT NNS NNS LNG0004 XCXXXXXCXX X1CX5CX2 NNS TGC NNS NNS NNS NNS NNS TGT NNS NNS LNG0005 XCXXXXXXCX X1CX6CX1 NNS TGC NNS NNS NNS NNS NNS NNS TGT NNS LNG0006 CXXXXXXXCX CX7CX1 TGC NNS NNS NNS NNS NNS NNS NNS TGT NNS LNG0007 CXXXXXXXXC CX8C TGC NNS NNS NNS NNS NNS NNS NNS NNS TGT LNG0008 CXXXXXXXXXC CX9C TGC NNS NNS NNS NNS NNS NNS NNS NNS NNS TGT LNG0009 CXXXXXXXXXXC CX10C TGC NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS TGT LNG0010 XXXXXXCXXCXXXXXX X6CX2CX6 NNS NNS NNS NNS NNS NNS TGC NNS NNS TGT NNS NNS KNS NNS NNS NNS LNG0011 XXXXXCXXXCXXXXXX X5CX3CX6 NNS NNS NNS NNS NNS TGC NNS NNS NNS TGT NNS NNS KNS NNS NNS NNS LNG0012 XXXXXCXXXXCXXXXX X5CX4CX5 NNS NNS NNS NNS NNS TGC NNS NNS NNS NNS TGT NNS KNS NNS NNS NNS LNG0013 XXXXCXXXXXCXXXXX x4CX5CX5 NNS NNS NNS NNS TGC NNS NNS NNS NNS NNS TGT NNS KNS NNS NNS NNS LNG0014 XXXXCXXXXXXCXXXX X4CX6Cx4 NNS NNS NNS NNS TGC NNS NNS NNS NNS NNS NNS TGT NNS NNS NNS NNS LNG0015 XXXCXXXXXXXCXXXX X3CX7Cx4 NNS NNS NNS TGC NNS NNS NNS NNS NNS NNS NNS TGT NNS NNS NNS NNS LNG0016 XXXCXXXXXXXXCXXX X3CX8CX3 NNS NNS NNS TGC NNS NNS NNS NNS NNS NNS NNS NNS TGT NNS NNS NNS LNG0017 XXCXXXXXXXXXCXXX X2CX9CX3 NNS NNS TGC NNS NNS NNS NNS NNS NNS NNS NNS NNS TGT NNS NNS NNS LNG0018 XXCXXXXXXXXXXCXX X2CX10CX2 NNS NNS TGC NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS TGT NNS NNS - The libraries were panned agains a series of therapeutically relevant targets using the following protocol: Wells on immunosorbent ELISA plates were coated with 5 μg/ml of the target antigen in PBS overnight at 4° C. Coated plates were washed with PBS, and non-specific sites were blocked with Blocking Buffer (PBS containing either 0.5% BSA or 0.5% Ovalbumin) for 2 h at room temperature. The plates were then washed with PBST (PBS containing 0.05% Tween 20), and phage particles at 1-5×1012/ml in Binding Buffer (Blocking Buffer containing 0.05% Tween 20) were added to the wells and incubated with shaking for 2 h at room temperature. Wells were then emptied and washed with PBST. Bound phage particles were eluted from the wells by incubation with 100 mM HCl for 10 min at room temperature, transferred to sterile tubes, and neutralized with 1M TRIS base. For infection, log phase E. Coli SS320 growing in Super Broth supplemented with 5 μg/ml Tetracycline were added to the neutralized phage eluate, and the culture was incubated with shaking for 30 min at 37° C. Infected cultures were then transferred to larger tubes containing Super Broth with 5 μg/ml Tetracycline and the cultures were incubated with shaking overnight at 37° C. The overnight cultures were cleared of E. Coli by centrifugation, and phage were precipitated from the supernatant following the addition of a solution of 20% PEG and 2.5MNaCl to a final PEG concentration of 4%. Precipitated phage were harvested by centrifugation, and the phage pellet was resuspended in 1 ml PBS, cleared of residual E. Coli by centrifugation, and transferred to a fresh tube. Phage concentrations were estimated spectrophotometrically and phage was utilized for the next round of selection. Individual clones were screened for target binding affinity after 3 or 4 rounds of phage panning. Individual plaques from phage clones selected during the panning were picked into Super Broth containing 5 μg/ml Tetracycline and grown overnight with shaking at 37° C. ELISA plates were prepared by coating antigen and control proteins (BSA, Ovalbumin, IgG) at 3 μg/ml in PBS overnight at 4° C. The plates were washed with PBS, and blocked with Blocking Buffer (PBS containing 0.5% BSA) for 2 h at room temperature. Overnight cultures were cleared of E. coli by centrifugation and the supernatant was diluted 1:10 in Binding Buffer (Blocking Buffer containing 0.05% Tween 20) and transferred to the ELISA plates after washing with PBST (PBS containing 0.05% Tween 20). The plates were incubated with shaking for 2 h at room temperature. Following washing with PBST, anti-M13-HRP (Pharmacia), 1:5000 dilution in PBS, was added to wells. The plates were incubated with shaking for 30 min at room temperature and washed with PBST, followed by PBS. A substrate solution containing 0.4 mg/ml ABTS and 0.001% H2O2 in 50 mM phosphate-citrate buffer was added to the wells, and allowed to develop for 40 min after which the plates were read in a plate reader at 405 nm. These ELISA readings allowed the determination of clone specificity, and antigen-specific clones were sequenced commercially via established methods.
-
TABLE X Sequences of EpCAM-specific binding modules S Y I C H N C L L S sNG0017S3.021 L R C W G M L C Y A sNG0017S3.017 L R C I G Q I C W R sNG0017S3.022 L K C L Y N I C W V sNG0017S3.024 R P G M A C S G Q L C W L N S P sNG001SS3.015 P H A L Q C Y G S L C W P S H L sNG001SS3.O18 R A G I T C H G H L C W P I T D sNG001SS3.019 R P A L K C I G T L C S L A N P sNG001SS3.014 P H G L W C H G S L C H Y P L A sNG001SS3.012 P H G L I C A G S I C F W P P P sNG001SS3.007 P R N L T C Y G Q I C F Q S Q H sNG001SS3.011 P H N L A C Q N S I C V R L P R sNG001SS3.021 P H G L T C T N Q I C F Y G N T sNG001SS3.006 L F C W G N V C H F sNG0017S3.006 L T C W G Q V C F R sNG0017S3.009 R C P S R V P W C V sNG0017S3.O11 Q L V C G F S D S S R L C Y M R sNG001SS3.009 L L C Y I T S P G N R L C S P Y sNG001SS3.022 -
TABLE X Sequences of VEGF-specific binding modules W E C T Q H W C P S sNG0025S3.021 A P F F S C S F G F C R D L Q T sNG0026S3.035 T P Y F R C Q F G F C F D S F S sNG0026S3.045 N P F F Y C V A G K C V D A P L sNG0026S3.029 D M R F L C R H G K C H D L P L sNG0026S3.034 P P F F V C S L G K C R D A H L sNG0026S3.043 P P Q F Q C V R G K C F D L T F sNG0026S3.053 I S T F F C S N G S C V D V P A sNG0026S3.006 P P H F R C F N G S C V D L S R sNG0026S3.051 N V H F W C H N H K C H D L V S sNG0026S3.040 L F F K C D V G H G C Y D I K H sNG0026S3.038 L Y F Q C F P N R G C S T L Q P sNG0026S3.002 P S F F C S P L L G C R D S L S sNG0026S3.052 G T P R C N P F R Q F C A I P S sNG0026S3.032 L C L P L G R W C P sNG0025S3.016 T S P A C N P F R H F C T L P T sNG0026S3.058 Q P P I C N P F R Q L C G I P L sNG0026S3.046 V H T F C N P F R Q M C S L P M sNG0026S3.027 R M V N C N P F N S W C S L P S sNG0026S3.001 S K H M C N P F H S W C G V P L sNG0026S3.047 R W P V C N P F L G Y C G I P N sNG0026S3.056 S K P T C N V F N S W C S V P L sNG0026S3.059 R P P A C N L F L S W C S Y D S sNG0026S3.004 G R S V C N P Y K S W C P V R Q sNG0026S3.O11 A S S C K D S P H F R C L F P L sNG0026S3.055 L A N C P N S P G F L C L H A V sNG0026S3.024 P F A C P H S S G F R C L Y N I sNG0026S3.005 S F T C S L F P S P H C T T L R sNG0026S3.054 L R L C T Y G G G K Y D C S S T sNG0026S3.050 G S Y C Q Y R P F S S F C N R S sNG0026S3.048 C S Y N Q V L G R A C sNG0025S3.001 P H C R Q H P L D R W M C S P S sNG0026S3.057 S L C S M F G D T P H W N C V P sNG0026S3.007 S S C S L F N N T R H W S C T D sNG0026S3.008 -
TABLE X Sequences of CD28-specific binding modules T T A Y P D C F W C S L F G P P sNG0028S3.085 M L D T T I C P W C S L F G P V sNG0028S3.081 M L X T T I C P W C S L F G P V sNG0028S3.018 E L L L E R C S W C S L F G P P sNG0028S3.086 S L S Q Q S C D W C W L F G P P sNG0028S3.060 K R L L E C G A L C A L F G P P sNG0028S3.008 H T I L T C D S G F C T L F G P sNG0028S3.012 N L W H V C H T S L C H S R L A sNG0028S3.092 N S F Y L C H S S V C G Q L P S sNG0028S3.082 A G F S C E N Y F F C P P K N L sNG0028S3.016 S W C T V F G N H D P S C N S R sNG0028S3.004 C S S N G R W K A H C sNG0028S3.076 L P N M W R V V V P D V Y D R R sNG0028S3.068 -
TABLE X Sequences of CD28-specific binding modules K H Y C F G P K S W T T C A R G sNG0030S3.096 P W C H L C P G S P S R C C Q P sNG0030S3.091 P E S K L I S E E D L N G D V S sNG0030S3.042 -
TABLE X Sequences of Tiel-specific binding modules I W D R V C R M N T C H Q H S H sNG0032S3.096 P Y T I F C L H S S C R S S S S sNG0032S3.087 D W C L T G P N T L S F C P R R sNG0032S3.031 -
TABLE X Sequences of DR4-specific binding modules L S T W R C L H D V C W P P L K sNG0033S3.072 -
TABLE X Sequences of DR5-specific binding modules V Y L T Q C G A Q L C L K R T N sNG0034S3.039 P Y L T S C G D R V C L K R P P sNG0034S3.001 P Y L S R C G G R I C M H D R L sNG0034S3.026 L K L T P C S H G V C M H R L R sNG0034S3.087 Y Y L T N C P K G H C L R R V D sNG0034S3.080 L Y L H S C S R G I C L S P R V sNG0034S3.082 F S C Q S S F P G R R M C E L R sNG0034S3.040 H R C S A H G S S S S F C P G S sNG0034S3.029 -
TABLE X Sequences of TrkA-specific binding modules K T W D C R N S G H C V I T F K sNG0035S3.074 A T W D C R D H N F S C V R L S sNG0035S3.089 - Anti-EpCAM peptides were isolated from random peptide libraries that were generated according to Scholle, et al. [Scholle, M. D., et al. (2005) Comb Chem High Throughput Screen, 8: 545-51] The naïve peptide libraries displayed cysteine-constrained peptides with cysteines spaced by 4 to 10 random residues. After three rounds of affinity selection with the above libraries, several EpCAM specific peptide ligands (EpCam1) were isolated (Table X). The EpCam1 isolates have a conserved cysteine spacing of four amino acids (CXXXXC). EpCam1 peptide ligands were then softly randomized (except cysteine positions) with codons encoding 3-9 residues and moved into a phagemid vector. Phagemid libraries were subsequently affinity selected against EpCAM to isolate peptide ligands optimized for binding (Table X, EpCam2). EpCam2 ligands contain the conserved CXXXXC cysteine spacing. In addition, the majority of anti-EpCam sequences do not contain a lysine residue, which allows for conjugation to free amine groups outside of the binding sequences. Furthermore, anti-EpCam peptide ligands can be genetically fused to URP sequences (of any length) and multimerized using iterative dimerization. The resulting anti-EpCAM MURPs can be used to specifically target EpCAM with increased affinity over monomer sequences. An example of a tetramer EpCAM-URP amino acid sequence is shown in
FIG. 31 . This sequence contains only two lysine residues that are located in the N-terminal Flag-tag. The side chains of these lysine residues are particularly suitable for drug conjugation. -
TABLE X Anti-EpCam sequences Name Sequence EpCam 1 LRCWGMLCYA LRCIGQICWR LKCLYNICWV LFCWGNVCHF LTCWGQVCFR RPGMACSGQLCWLNSP PHALQCYGSLCWPSHL RAGITCHGHLCWPITD RPALKCIGTLCSLANP PHGLWCHGSLCHYPLA PHGLICAGSICFWPPP PRNLTCYGQICFQSQH PHNLACQNSICVRLPR PHGLTCTNQICFYGNT EpCam 2 HSLTCYGQICWVSNI PTLTCYNQVCWVNRT PALRCLGQLCWVTPT PGLRCLGTLCWVPNR RNLTCWNTVCYAYPN RGLKCLGQLCWVSSN PTLKCSGQICWVPPP RNLECLGNVCSLLNQ PTLTCLNNLCWVPPQ RGLKCSGHLCWVTPQ HGLTCHNTVCWVHHP HTLECLGNICWVINQ HGLTCYNQICWAPRP HGLACYNQLCWVNPH RGLACQGNICWRLNP RAITCLGTLCWPTSP LTLECIGNICYVPHH - Binding modules can be affinity matured, or lengthened, by the addition of URP-like linkers and random sequence to the N-terminus, C-terminus, or both N- and C-terminus of the binding sequence.
FIG. 32 shows the addition of naïve cysteine-constrained sequences to an anti-EpCAM binding module. Libraries of random sequence additions can be generated using a single-stranded or double-stranded DNA cloning approaches. Once generated, libraries can be affinity selected against the initial target protein or a second protein. For example, an addition library that contains an anti-EpCAM binding module can be used to select sequences that contain 2 or more binding sites to the target protein. - A series of oligonucleotides was designed to construct a library based on the VEGF-binding 1SS peptide FTCTNHWCPS. The oligonucleotides incorporate variations in cysteine distance patterns of the flanking sequences while the VEGF-binding peptide sequence was kept fixed.
-
Forward oligos: LMS70-1 CAGGCAGCGGGCCCGTCTGGCCCGTGYTTTACTTGTACGAATCATTGGTG TCCT LMS70-2 CAGGCAGCGGGCCCGTCTGGCCCGTGYNNKTTTACTTGTACGAATCATTG GTGTCCT LMS70-3 CAGGCAGCGGGCCCGTCTGGCCCGTGYNNKNNKTTTACTTGTACGAATCA TTGGTGTCCT LMS70-4 CAGGCAGCGGGCCCGTCTGGCCCGTGYNHTNHTNHTTTTACTTGTACGAA TCATTGGTGTCCT LMS70-5 CAGGCAGCGGGCCCGTCTGGCCCGTGYNHTNHTNHTNHTTTTACTTGTAC GAATCATTGGTGTCCT LMS70-6 CAGGCAGCGGGCCCGTCTGGCCCGTGYKMTKMTKMTKMTKMTTTTACTTG TACGAATCATTGGTGTCC Reverse oligos (reverse complemented): LMS70-1R ACCGGAACCACCAGACTGGCCRCACGAAGGACACCAATGATTCGTACAA LMS70-2R ACCGGAACCACCAGACTGGCCRCAMNNCGAAGGACACCAATGATTCGTAC AA LMS70-3R ACCGGAACCACCAGACTGGCCRCAMNNMNNCGAAGGACACCAATGATTCG TACAA LMS70-4R ACCGGAACCACCAGACTGGCCRCAADNADNADNCGAAGGACACCAATGAT TCGTACAA LMS70-5R ACCGGAACCACCAGACTGGCCRCAADNADNADNADNCGAAGGACACCAAT GATTCGTACAA LMS70-6R ACCGGAACCACCAGACTGGCCRCAAKMAKMAKMAKMAKMCGAAGGACACC AATGATTCGTACAA - Mixture 1 (from 100 μM stocks): 100 μl 70-6, 33 μl 70-5, 11 μl 70-4, 3.66 μl 70-3, 1.2 μl 70-2, 0.4 μl 70-1. Mixture 2 (from 100 μM stocks): 100 μl 70-6R, 33 μl 70-5R, 11 μl 70-4R, 3.66 μl 70-3R, 1.2 μl 70-2R, 0.4 μl 70-1R
- 10.0 μl Template Oligo (5 μM), 10.0 μl 10× Buffer, 2.0 dNTPs (10 mM), 1.0 μl cDNA Polymerase (Clonetech), 77
μl DS H 20. PCR program: 95° C. 1 min, (95° C. 15 sec, 54° C. 30 sec, 68° C. 15 sec)×5, 68° C. 1 min - Primers, 10.0 μl Assembled mixture, 10.0 μl 10× buffer, 2.0 dNTPs (10 mM), 10.0 μl LIBPTF (5 μM), 10.0 μl LIBPTR (5 μM), 1.0 μl cDNA polymerase (Clonetech), 57
μl DS H 20. PCR program: 95° C. 1 min, (95° C. 15 sec, 54° C. 30 sec, 68° C. 15 sec)×25, 68° C. 1 min. The product was purified by Amicon column Y10. The assembled product was digested with SfiI and BstXI and ligated into the phagemid vector pMP003. Ligation was performed over night at 16° C. in a MJ PCR machine. Ligation then was purified by EtOH precipitation. Transformation into fresh competent ER2738 cells by Electroporation. - The resulting library was panned against VEGF as described below. Several isolates were identified that showed improved binding to VEGF relative to the 1SS starting sequence. Binding and expression data are shown in
FIG. 38 . Sequences and results of Western analysis of buildup clones is shown inFIG. 39 . - First Round Panning:
- 1) First round,
coat 4 wells per library to be screened. Coat the well of a Costar 96-well ELISA plate with 0.25 μg of VEGF121 antigen in 25 μl of PBS. Cover the plate with a plate sealer. Coating can be performed overnight at 4° C. or for 1 h at 37° C. - 2) After shaking out the coating solution, block the well by adding 150 μl of PBS/
BSA 1%. Seal and incubate for 1 h at 37° C. - 3) After shaking out the blocking solution, add 50 μl of freshly prepared phage (see library reamplification protocol) to the well. For the first round only, also add 5 μl of Tween 5%. Seal the plate and incubate for 2 h at 37° C.
- In the meantime, inoculate 2 ml SB medium plus 2 μl of 5 mg/ml Tetracycline with 2 μl of an ER 2738 cell preparation and allow growth at 250 rpm and 37° C. for 2.5 h. Grow 1 culture for each library that is screened including negative selections. Take all precautions to avoid a contamination of the culture with phage.
- 4) Shake out the phage solution, add 150 μl of PBS/Tween 0.5% to the well and pipette 5 times vigorously up and down. Wait 5 min, shake out, and repeat this washing step. In the first round, wash in this fashion 5 times, in the
second round 10 times, and in the third, fourth andfifth round 15 times. - 5) After shaking out the final washing solution, add 50 μl of freshly prepared 10 mg/ml trypsin in PBS, seal, and incubate for 30 min at 37°
C. Pipette 10 times vigorously up and down and transfer the eluate (4×50 μl in the first round, 2×50 ml in the second round, 1×50 μl in the subsequent rounds) to the prepared 2-ml E. coli culture and incubate at room temperature for 15 min. - 6) Add 6 ml of pre-warmed SB medium, 1.6 μl of carbenicillin and 6 μl of 5 mg/ml Tetracycline. Transfer the culture into a 50-ml polypropylene tube.
- 7) Shake the 8-ml culture at 250 rpm and 37° C. for 1 h, add 2.4 μl 100 mg/ml carbenicillin, and shake for an additional hour at 250 rpm and 37° C.
- 8) Add 1 ml of VCSM13 helper phage and transfer to a 500-ml polypropylene centrifuge bottle. Add 91 ml of pre-warmed (37° C.) SB medium and 46 μL of 100 mg/ml carbenicillin and 92 μl of 5 mg/ml Tetracycline. Shake the 100-ml culture at 300 rpm and 37° C. for 1½ to 2 h.
- 9) Add 140 μl of 50 mg/ml kanamycin and continue shaking at 300 rpm and 37° C. overnight.
- 10) Spin at 4000 rpm for 15 min at 4° C. Transfer the supernatant to a clean 500-ml centrifuge bottle and add add 25 ml of 20% PEG-8000/NaCl 2.5M. Store on ice for 30 min.
- 11) Spin at 9000 rpm for 15 min at 4° C. Discard the supernatant, drain inverted on a paper towel for at least 10 min, and wipe off remaining liquid from the upper part of the centrifuge bottle with a paper towel.
- 12) Resuspend the phage pellet in 2 ml of PBS/BSA 0.5%/Tween 0.5% buffer by pipetting up and down along the side of the centrifuge bottle and transfer to a 2-ml microcentrifuge tube. Resuspend further by pipetting up and down using a 1-ml pipette tip, spin at full speed in a microcentrifuge for 1 min at 4° C., and pass the supernatant through a 0.2-μm filter into a sterile 2-ml microcentrifuge tube.
- 13) Continue from step 3) for the next round or store the phage preparation at 4° C. Sodium azide may be added to 0.02% (w/v) for long-term storage. Only freshly prepared phage should be used for each round.
- Second Round Panning
- Second round,
coat 2 wells per library to be screened. Coat the well of a Costar 96-well ELISA plate with 0.25 μg of VEGF121 antigen in 25 μl of PBS. Cover the plate with a plate sealer. Coating can be performed overnight at 4° C. or for 1 h at 37° C. - Also block 2 uncoated wells for each library to be used as negative control for the enrichment ratio calculation.
- Third Round Panning
- Third round,
coat 1 well per library to be screened. Coat the well of a Costar 96-well ELISA plate with 0.25 μg of VEGF12, antigen in 25 μl of PBS. Cover the plate with a plate sealer. Coating can be performed overnight at 4° C. or for 1 h at 37° C. - Also block 1 uncoated well for each library to be used as negative control for the enrichment ratio calculation.
- 1. Biotinylate the target protein according to manufacturer.
- 2. Coat a total of 8 wells (per selection) with 1.0 μg of neutravidin (Pierce) in PBS and incubate overnight at 4° C.
- 3. Block the wells with SuperBlock (Pierce) for 1 h at room temp. Store plate with blocking buffer until needed (in Step 6).
- 4. Use 100 nM of biotinylated target protein and add 1012 phage/ml (in PBST) for a total volume of 100-200 μl using SuperBlock plus
Tween 20 0.05%. - 5. Tumble phage-target mixture at room temp for at least 1 h.
- 6. Dilute 100 μl phage-target mix with 700 μl SuperBlock, mix, and add 100 μl to each of 8 neutravidin-coated wells (from Step 3).
- 7. Incubate for 5 min at room temp.
- 8.
Wash 8× with PBST. - 9. Elute phage with 100 μl of 100 mM HCl for 10 min.
- 10. Neutralize by adding 10 μl of 1M TRIS pH=8.0.
- 11. Infect cells for plating or amplify phage for a subsequent round of solution panning.
- 1) Add 0.5 ml SB containing 50 μg/ml carbenicillin to 96 deep well plate. Pick one colony and inoculate wells.
- 2) Shake the plate containing the bacterial cultures at 300 rpm o/n at 37° C.
- 3) Prepare 4 ng/μl target protein solution in PBS. Add 25 μl (100 ng) of protein to each well and incubate overnight at 4° C.
- 4) Shake out coated ELISA plates and wash 2× with PBS. Add 150 μl/well PBS+0.5% BSA (blocking buffer). Block for 1 at RT.
- 5) Spin down microtube racks (3000 rpm; 20 min).
- 6) Prepare binding buffer (blocking buffer+0.5% Tween 20). Aliquot 135 μl binding buffer per well in low protein-binding 96 well plate.
- 7) Shake out wells on ELISA plates and wash 2 times with PBST (PBS+0.5% Tween 20).
- 8) Dilute 15 μl phage from o/n cultures 1:10 in PBST, mix by pipetting, and transfer 30 μl to each protein-coated well. Incubate 2 h at RT with gentle shaking.
- 9) Wash plates 6 times with PBST.
- 10) Add 50 μl antiM13-HRP 1:5000 in binding buffer to the wells.
Incubate 30 min with gentle shaking at RT. - 11) Wash the
plates 4 times with PBST, followed by 2 times with H2O. - 12) Prepare 6 ml of ABTS solution (5.88 ml of citrate buffer plus 120 μl ABTS and 2 μl H2O2).
Aliquot 50 μl per well on each ELISA plate - 13) Incubate at RT and read O.D. at 405 nm using an ELISA plate reader at appropriate time points depending on the signal (up to 1 h)
- Phage displayed libraries of 10e9 to 10e11 cyclic peptides with 4, 5, 6, 7, 8, 9, 10, 11 and 12 randomized or partially randomized amino acids between the disulfide-bonded cystines, and in some cases additional randomized amino acids on the outside of the cystine pair, were created by standard methods. Panning of these cyclic peptide libraries against a number of targets, including human VEGF, reliably yielded peptides that bound specifically to hVEGF and not to BSA, Ovalbumin or IgG.
- Two libraries were designed based on the Plexin scaffold. The Pfam protein database was used for phylogenetic alignment of naturally occurring plexin domains as shown in
FIG. 35 . The middle part of plexin scaffold (Cys24-Gly25-Trp26-Cys27) is conserved in both library designs and served as a crossover region for N- and C-library generation. The randomization schemes of both plexin libraries are shown inFIG. 36 . The two libraries were generated by overlapping two library-encoding oligos at the crossover region and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) and cloning into phagemid vector pMP003. The resulting plexin libraries were designated LMP031 (N terminal library) and LMP032 (C terminal library) and each was represented by a complexity of approximately 5×108 independent transformants. For validation, approximately 24 Carb-resistant clones from each unselected library were analyzed by PCR. Clones that gave a correct size fragment (375 bp) were further analyzed by DNA sequencing. Correct full-length plexin sequences were obtained for 50% and 67% of clones derived from LMP031 and LMP032 libraries, respectively. - The two libraries were mixed together at 50/50 ratio and panned in parallel against VEGF, death receptor Dr4, ErbB2, and HGFR immobilized on 96-well ELISA plates. Four rounds of panning were carried out using 1000 ng of protein target in the first round, 500 ng in the second round, 250 ng in the third round, and 100 ng in the fourth round. After the final round of panning, 192 Carb-resistant clones from each selection were analyzed for binding to 100 ng immobilized protein target, human IgG, Ovalbumin, and BSA by phage ELISA using polyclonal anti-M13 Ab conjugated to horseradish peroxidase for detection. The highest percentage of positive clones was obtained for target DR4 (69%), followed by target ErbB2 (53%), HGFR (13%), and BoNT target (1%). Positive clones were further analyzed by PCR and by DNA sequencing. All clones revealed unique sequences and all but one (against DR4) were derived from LMP032 (C terminal library). Sequences of some of the identified target-selective isolates are shown in
FIG. 37 . - For further analysis, an assortment of selected target-specific binders are first subcloned into protein expression vector pVS001, then produced as soluble microproteins, and finally purified by heat lysis. The purified target-specific microproteins are analysed by protein ELISA to confirm the target recognition, by SDS-PAGE to confirm monomer formation, and by surface plasmon resonance to measure their affinities to target. The best clones are used in the next round of library generation to further improve their properties.
- Phage displayed libraries of 10e8 to 10e10 of 3 finger toxin (3FT) scaffolds with partially randomized amino acids of
fingertip 1 and descending part offinger 2 orfingertip 3 and ascending part offinger 2 were created by standard methods. - Two 3FT scaffolds were used as a template for 3FT library generation (
fingers FIG. 33 . A library was designed such that two surface loops of the toxin are randomized as illustrated inFIG. 34 . The library of partially randomized 3FT scaffold was generated by overlapping four library-encoding oligos at the annealing regions and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) into phagemid vector pMP003. The resulting 3FT library was designated LMP041. - The aim here is to use the peptides that have been identified to be specific for target of interest in order to generate 3SSplus target-specific binders. This strategy is illustrated by using VEGF-specific peptide transfer into
fingertip 1 of 3FT scaffold and by modifying the AA residues offinger 2, which are in close proximity from target specific sequence to generate high affinity VEGF binders. Phage displayed libraries of 10e8 to 10e10 of 3 finger toxin (3FT) scaffolds with VEGF specific sequence offingertip 1 and partially randomized descending part offinger 2 was created by standard methods as described in example above except 2random finger 1 forward primers were replaced by F1-VEGF-specific forward primer encoding the following sequence: P S G P S C H T T N H W P I S A V T C P P. - The focused (VEGF-specific) 3FT scaffold library with partially randomized
finger 2 was generated by overlapping four library-encoding oligos at the annealing regions and using pull-thru PCR followed by restriction cloning (SfiI/BstXI) into phagemid vector pMP003. The resulting 3FT library was designated LMP042. - The plasma half-life of MURPs can be measured after i.v. or i.p. injection of the MURP into catheterized rats essentially as described by [Pepinsky, R. B., et al. (2001) J Pharmacol Exp Ther, 297: 1059-66]. Blood samples can be withdrawn at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 5 h, 1 d, 2 d, 3 d) and the plasma concentration of the MURP can be measured using ELISA. Pharmacokinetic parameters can be calculated using WinNonlin version 2.0 (Scientific Consulting Inc., Apex, N.C.). To analyze the effect of the URP module one can compare on plasma half-life of a protein containing the URP module with the plasma half-life of the same protein lacking the URP module.
- Solubility of MURPs can be determined by concentrating purified samples of MURPs in physiological buffers like phosphate buffered saline to various concentrations in the range of 0.01 mg/ml to 10 mg/ml. Samples can be incubated for up to several weeks. Samples where the concentration exceeds the solubility of the MURP show precipitation as indicated by turbidity, which can be measured in an absorbance reader. On can remove precipitated material by centrifugation or filtration and measure the concentration of remaining protein in the supernatant using a protein assay like the Bradford assay of by measuring the absorbance at 280 nm. Solubility studies can be accelerated by freezing the samples at −20 C and subsequent thawing. This process frequently leads to the precipitation of poorly soluble proteins.
- One can coat MURPs of interest into microtiter plates and control proteins in other wells of the plate. Subsequently, one can add serum samples of interest to the wells for 1 hour. Subsequently, the wells can be washed with a plate washer. Bound serum proteins can be detected by adding antibodies against serum proteins that have been conjugated with enzymes like horse radish peroxidase or alkaline phosphatase for detection. Another way to detec serum binding to MURPs to add the MURP of interest to serum for about 1 hour to allow binding. Subsequently, one can immunoprecipitate the MURP using an antibody against an epitope in the MURP sequence. The precipitated samples can be analyzed by PAGE and optionally by Western to detect any proteins that co-precipitated with the MURP. One can identify the serum proteins that show co-precipitation by mass spectrometry.
Claims (17)
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/634,556 US20100189682A1 (en) | 2005-09-27 | 2009-12-09 | Biologically active proteins having increased In Vivo and/or In Vitro stability |
US13/396,403 US20120220011A1 (en) | 2005-09-27 | 2012-02-14 | Unstructured recombinant polymers and uses thereof |
US13/439,738 US20120230947A1 (en) | 2005-09-27 | 2012-04-04 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/102,297 US20140356326A1 (en) | 2005-09-27 | 2013-12-10 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/970,723 US9938331B2 (en) | 2005-09-27 | 2015-12-16 | Biologically active proteins having increased in vivo and/or in vitro stability |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72127005P | 2005-09-27 | 2005-09-27 | |
US72118805P | 2005-09-27 | 2005-09-27 | |
US74341006P | 2006-03-06 | 2006-03-06 | |
US74362206P | 2006-03-21 | 2006-03-21 | |
US11/528,927 US20070191272A1 (en) | 2005-09-27 | 2006-09-27 | Proteinaceous pharmaceuticals and uses thereof |
US11/528,950 US20070212703A1 (en) | 2005-09-27 | 2006-09-27 | Proteinaceous pharmaceuticals and uses thereof |
US11/715,276 US7855279B2 (en) | 2005-09-27 | 2007-03-06 | Unstructured recombinant polymers and uses thereof |
US12/634,556 US20100189682A1 (en) | 2005-09-27 | 2009-12-09 | Biologically active proteins having increased In Vivo and/or In Vitro stability |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/528,927 Continuation-In-Part US20070191272A1 (en) | 2005-09-27 | 2006-09-27 | Proteinaceous pharmaceuticals and uses thereof |
US11/715,276 Continuation US7855279B2 (en) | 2005-09-27 | 2007-03-06 | Unstructured recombinant polymers and uses thereof |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/396,403 Continuation US20120220011A1 (en) | 2005-09-27 | 2012-02-14 | Unstructured recombinant polymers and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20100189682A1 true US20100189682A1 (en) | 2010-07-29 |
Family
ID=46328572
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/715,276 Active 2027-08-22 US7855279B2 (en) | 2005-09-27 | 2007-03-06 | Unstructured recombinant polymers and uses thereof |
US12/634,556 Abandoned US20100189682A1 (en) | 2005-09-27 | 2009-12-09 | Biologically active proteins having increased In Vivo and/or In Vitro stability |
US12/939,120 Active 2026-10-23 US8492530B2 (en) | 2005-09-27 | 2010-11-03 | Unstructured recombinant polymers and compositions comprising same |
US13/396,403 Abandoned US20120220011A1 (en) | 2005-09-27 | 2012-02-14 | Unstructured recombinant polymers and uses thereof |
US13/439,738 Abandoned US20120230947A1 (en) | 2005-09-27 | 2012-04-04 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/102,297 Abandoned US20140356326A1 (en) | 2005-09-27 | 2013-12-10 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/970,723 Active US9938331B2 (en) | 2005-09-27 | 2015-12-16 | Biologically active proteins having increased in vivo and/or in vitro stability |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/715,276 Active 2027-08-22 US7855279B2 (en) | 2005-09-27 | 2007-03-06 | Unstructured recombinant polymers and uses thereof |
Family Applications After (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/939,120 Active 2026-10-23 US8492530B2 (en) | 2005-09-27 | 2010-11-03 | Unstructured recombinant polymers and compositions comprising same |
US13/396,403 Abandoned US20120220011A1 (en) | 2005-09-27 | 2012-02-14 | Unstructured recombinant polymers and uses thereof |
US13/439,738 Abandoned US20120230947A1 (en) | 2005-09-27 | 2012-04-04 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/102,297 Abandoned US20140356326A1 (en) | 2005-09-27 | 2013-12-10 | Biologically active proteins having increased in vivo and/or in vitro stability |
US14/970,723 Active US9938331B2 (en) | 2005-09-27 | 2015-12-16 | Biologically active proteins having increased in vivo and/or in vitro stability |
Country Status (1)
Country | Link |
---|---|
US (7) | US7855279B2 (en) |
Cited By (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080286808A1 (en) * | 2005-09-27 | 2008-11-20 | Volker Schellenberger | Methods for production of unstructured recombinant polymers and uses thereof |
US20090092582A1 (en) * | 2007-08-15 | 2009-04-09 | Oren Bogin | Compositions and methods for modifying properties of biologically active polypeptides |
US20090099031A1 (en) * | 2005-09-27 | 2009-04-16 | Stemmer Willem P | Genetic package and uses thereof |
US20100268055A1 (en) * | 2007-07-19 | 2010-10-21 | Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University | Self-Anchoring MEMS Intrafascicular Neural Electrode |
US20100323956A1 (en) * | 2009-02-03 | 2010-12-23 | Amunix, Inc. | Glucose-regulating polypeptides and methods of making and using same |
US20110046061A1 (en) * | 2009-02-03 | 2011-02-24 | Amunix Operating, Inc. | Coagulation factor VII compositions and methods of making and using same |
US20110077199A1 (en) * | 2009-02-03 | 2011-03-31 | Amunix, Inc. | Growth hormone polypeptides and methods of making and using same |
US20110172146A1 (en) * | 2009-02-03 | 2011-07-14 | Amunix Operating, Inc. | Growth hormone polypeptides and methods of making and using same |
US20110171687A1 (en) * | 2005-09-27 | 2011-07-14 | Amunix Operating, Inc. | Unstructured recombinant polymers and compositions comprising same |
US20120207743A1 (en) * | 2011-02-14 | 2012-08-16 | Allergan, Inc. | Inhibiting Aberrant Blood Vessel Formation Using Retargeted Endopeptidases |
WO2012142142A2 (en) * | 2011-04-12 | 2012-10-18 | Temple University - Of The Commonwealth System Higher Education | Adiponectin receptor agonists and methods of use |
US8557961B2 (en) | 2010-04-02 | 2013-10-15 | Amunix Operating Inc. | Alpha 1-antitrypsin compositions and methods of making and using same |
US8673860B2 (en) | 2009-02-03 | 2014-03-18 | Amunix Operating Inc. | Extended recombinant polypeptides and compositions comprising same |
US9050318B2 (en) | 2009-12-06 | 2015-06-09 | Biogen Idec Hemophilia Inc. | Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof |
WO2015195531A2 (en) | 2014-06-18 | 2015-12-23 | Albert Einstein College Of Medicine, Inc. | Syntac polypeptides and uses thereof |
US9233145B2 (en) | 2010-07-09 | 2016-01-12 | Biogen Hemophila Inc. | Factor IX polypeptides and methods of use thereof |
US9486507B2 (en) | 2011-06-10 | 2016-11-08 | Biogen Ma Inc. | Pro-coagulant compounds and methods of use thereof |
WO2017201210A1 (en) | 2016-05-18 | 2017-11-23 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
WO2017201131A1 (en) | 2016-05-18 | 2017-11-23 | Albert Einstein College Of Medicine, Inc. | Variant pd-l1 polypeptides, t-cell modulatory multimeric polypeptides, and methods of use thereof |
US9849188B2 (en) | 2009-06-08 | 2017-12-26 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
US10010622B2 (en) | 2011-07-08 | 2018-07-03 | Bioverativ Therapeutics Inc. | Factor VIII chimeric and hybrid polypeptides, and methods of use thereof |
WO2018129474A1 (en) | 2017-01-09 | 2018-07-12 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
US10023628B2 (en) | 2012-07-06 | 2018-07-17 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor VIII polypeptides and uses thereof |
US10172953B2 (en) | 2012-02-27 | 2019-01-08 | Amunix Operating Inc. | XTEN conjugate compositions and methods of making same |
WO2019051127A1 (en) | 2017-09-07 | 2019-03-14 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptide with conjugation sites and methods of use thereof |
WO2019139896A1 (en) | 2018-01-09 | 2019-07-18 | Cue Biopharma, Inc. | Multimeric t-cell modulatory polypeptides and methods of use thereof |
US10370430B2 (en) | 2012-02-15 | 2019-08-06 | Bioverativ Therapeutics Inc. | Recombinant factor VIII proteins |
US10421798B2 (en) | 2012-02-15 | 2019-09-24 | Bioverativ Therapeutics Inc. | Factor VIII compositions and methods of making and using same |
US10548953B2 (en) | 2013-08-14 | 2020-02-04 | Bioverativ Therapeutics Inc. | Factor VIII-XTEN fusions and uses thereof |
US10584147B2 (en) | 2013-11-08 | 2020-03-10 | Biovertiv Therapeutics Inc. | Procoagulant fusion compound |
US10588949B2 (en) | 2013-03-15 | 2020-03-17 | Bioverativ Therapeutics Inc. | Factor IX polypeptide formulations |
US10656167B2 (en) | 2011-07-25 | 2020-05-19 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
WO2020132297A1 (en) | 2018-12-19 | 2020-06-25 | Cue Biopharma, Inc. | Multimeric t-cell modulatory polypeptides and methods of use thereof |
US10745680B2 (en) | 2015-08-03 | 2020-08-18 | Bioverativ Therapeutics Inc. | Factor IX fusion proteins and methods of making and using same |
WO2020181062A1 (en) | 2019-03-06 | 2020-09-10 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
US10988522B2 (en) | 2016-02-02 | 2021-04-27 | University Of Southern California | Proteolically resistant cyclotides with angiotensin 1-7 like activity |
US11225650B2 (en) | 2012-09-25 | 2022-01-18 | Bioverativ Therapeutics Inc. | Methods of using FIX polypeptides |
WO2022032187A1 (en) | 2020-08-07 | 2022-02-10 | Bristol-Myers Squibb Company | Fgf21 combined with ccr2/5 antagonists for the treatment of fibrosis |
WO2022115597A1 (en) | 2020-11-25 | 2022-06-02 | Bristol-Myers Squibb Company | Methods of treating liver diseases |
US11370827B2 (en) * | 2012-01-12 | 2022-06-28 | Bioverativ Therapeutics Inc. | Chimeric factor VIII polypeptides and uses thereof |
US11713358B2 (en) | 2015-08-28 | 2023-08-01 | Amunix Pharmaceuticals, Inc. | Chimeric polypeptide assembly and methods of making and using the same |
EP4360647A2 (en) | 2016-12-22 | 2024-05-01 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
US12030925B2 (en) | 2018-05-18 | 2024-07-09 | Bioverativ Therapeutics Inc. | Methods of treating hemophilia A |
Families Citing this family (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1929073A4 (en) * | 2005-09-27 | 2010-03-10 | Amunix Inc | Proteinaceous pharmaceuticals and uses thereof |
DK1996220T4 (en) † | 2006-03-06 | 2023-11-13 | Amunix Operating Inc | UNSTRUCTURED RECOMBINANT POLYMERS AND THEIR USES |
WO2008121563A2 (en) | 2007-03-30 | 2008-10-09 | Ambrx, Inc. | Modified fgf-21 polypeptides and their uses |
DE602008005596D1 (en) * | 2007-06-21 | 2011-04-28 | Univ Muenchen Tech | BIOLOGICALLY ACTIVE PROTEINS WITH INCREASED IN VIVO AND / OR IN VITRO STABILITY |
ES2674567T3 (en) * | 2010-05-21 | 2018-07-02 | Merrimack Pharmaceuticals, Inc. | Bispecific Fusion Proteins |
EP2680879B1 (en) * | 2011-03-04 | 2021-01-06 | Shire Human Genetic Therapies, Inc. | Peptide linkers for polypeptide compositions and methods for using same |
WO2012174446A1 (en) | 2011-06-17 | 2012-12-20 | President And Fellows Of Harvard College | Frizzled 2 as a target for therapeutic antibodies in the treatment of cancer |
CN110763842A (en) | 2011-06-29 | 2020-02-07 | 中央研究院 | Capture, purification and release of biological substances using surface coatings |
WO2013067133A1 (en) * | 2011-11-04 | 2013-05-10 | The Texas A & M University System | Production of tsg-6 protein |
WO2013185114A2 (en) | 2012-06-08 | 2013-12-12 | Biogen Idec Ma Inc. | Chimeric clotting factors |
EP4079316A1 (en) | 2012-06-08 | 2022-10-26 | Bioverativ Therapeutics Inc. | Procoagulant compounds |
US9221888B2 (en) | 2012-11-13 | 2015-12-29 | University of Pittsburgh—of the Commonwealth System of Higher Education | Bone substitute nanocomposites and methods of synthesis using multiphosphorylated peptides |
WO2014100913A1 (en) * | 2012-12-24 | 2014-07-03 | Beijing Anxinhuaide Biotech. Co., Ltd | Improving the half life of a therapeutic polypeptide by fusing with a trimeric scaffold protein via a spacer |
WO2014116865A1 (en) * | 2013-01-24 | 2014-07-31 | Mayo Foundation For Medical Education And Research | Methods and materials for detecting c9orf72 hexanucleotide repeat expansion positive frontotemporal lobar degeneration or c9orf72 hexanucleotide repeat expansion positive amyotrophic lateral sclerosis |
WO2014183109A1 (en) * | 2013-05-10 | 2014-11-13 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Compositions and methods for simultaneous detection of hcv antigen/antibody |
US9469677B2 (en) | 2013-11-13 | 2016-10-18 | University of Pittsburgh—of the Commonwealth System of Higher Education | Biomimetic coating of magnesium alloy for enhanced corrosion resistance and calcium phosphate deposition |
TW201623605A (en) | 2014-04-01 | 2016-07-01 | 中央研究院 | Methods and systems for cancer diagnosis and prognosis |
US10112198B2 (en) | 2014-08-26 | 2018-10-30 | Academia Sinica | Collector architecture layout design |
NZ730563A (en) | 2014-10-14 | 2019-05-31 | Halozyme Inc | Compositions of adenosine deaminase-2 (ada2), variants thereof and methods of using same |
MA40835A (en) | 2014-10-23 | 2017-08-29 | Biogen Ma Inc | ANTI-GPIIB / IIIA ANTIBODIES AND THEIR USES |
MX2017004947A (en) | 2014-10-24 | 2017-06-29 | Bristol Myers Squibb Co | Modified fgf-21 polypeptides and uses thereof. |
MA40861A (en) | 2014-10-31 | 2017-09-05 | Biogen Ma Inc | ANTI-GLYCOPROTEIN IIB / IIIA ANTIBODIES |
BR112017009951A2 (en) * | 2014-11-11 | 2017-12-26 | Amunix Operating Inc | directed xten conjugated compositions and methods of their production |
EP3303380B1 (en) | 2015-06-02 | 2020-01-15 | Novo Nordisk A/S | Insulins with polar recombinant extensions |
MA43348A (en) | 2015-10-01 | 2018-08-08 | Novo Nordisk As | PROTEIN CONJUGATES |
EP3865147A1 (en) | 2015-10-02 | 2021-08-18 | Silver Creek Pharmaceuticals, Inc. | Bi-specific therapeutic proteins for tissue repair |
CN106749661B (en) * | 2015-11-13 | 2021-05-14 | 北京大学 | anti-PSMP monoclonal antibody and application thereof |
US10107726B2 (en) | 2016-03-16 | 2018-10-23 | Cellmax, Ltd. | Collection of suspended cells using a transferable membrane |
JP6174778B1 (en) * | 2016-12-19 | 2017-08-02 | シミックホールディングス株式会社 | Liver-type fatty acid binding protein preparation, method for evaluating the preparation, method for suppressing the fluctuation range of the measurement value caused by liver-type fatty acid binding protein in the measurement using the preparation, calibration curve for liver-type fatty acid binding protein Method for preparing and method for quantifying the protein |
CN106819380A (en) * | 2016-12-31 | 2017-06-13 | 新昌县田野泉养殖技术开发有限公司 | Biological protein feed and preparation method thereof, application method |
EP3592758A4 (en) * | 2017-03-06 | 2021-04-14 | Synvitrobio, Inc. | Methods and systems for cell-free biodiscovery of natural products |
US11471537B2 (en) | 2017-04-05 | 2022-10-18 | Novo Nordisk A/S | Oligomer extended insulin-Fc conjugates |
AR111597A1 (en) * | 2017-06-19 | 2019-07-31 | Swedish Orpham Biovitrum Ab Publ | FUSION PROTEIN WITH MIDDLE LIFE EXTENSION POLYPEPTIDE |
WO2019028040A1 (en) * | 2017-07-31 | 2019-02-07 | Vasculonics Llc | Modified ddah polypeptides comprising a pharmacokinetic enhancing moiety, improved pharmacology and their uses |
WO2019201866A1 (en) * | 2018-04-16 | 2019-10-24 | Swedish Orphan Biovitrum Ab (Publ) | Fusion protein |
MA54070A (en) | 2018-10-29 | 2021-09-08 | Biogen Ma Inc | HUMANIZED AND STABILIZED FC5 VARIANTS FOR ENHANCED TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER |
EP3921335A4 (en) * | 2019-02-08 | 2023-02-08 | The Regents Of The University Of California | Compositions and methods involving layilin |
EP4085077A4 (en) | 2019-12-31 | 2024-01-17 | Beijing QL Biopharmaceutical Co., Ltd. | Fusion proteins of glp-1 and gdf15 and conjugates thereof |
WO2021139744A1 (en) | 2020-01-11 | 2021-07-15 | Beijing Ql Biopharmaceutical Co., Ltd. | Conjugates of fusion proteins of glp-1 and fgf21 |
GB202004514D0 (en) | 2020-03-27 | 2020-05-13 | Inst De Medicina Molecular Joaeo Lobo Antunes | Treatment of Immunosuppressive Cancer |
WO2022195629A1 (en) * | 2021-03-18 | 2022-09-22 | National Institute Of Pharmaceutical Education And Research (Niper) | Engineered arginase constructs, method of generation and uses thereof |
WO2023212293A1 (en) | 2022-04-29 | 2023-11-02 | Broadwing Bio Llc | Complement factor h related 4-specific antibodies and uses thereof |
WO2023212294A1 (en) | 2022-04-29 | 2023-11-02 | Broadwing Bio Llc | Angiopoietin-related protein 7-specific antibodies and uses thereof |
WO2023212298A1 (en) | 2022-04-29 | 2023-11-02 | Broadwing Bio Llc | Bispecific antibodies and methods of treating ocular disease |
Citations (88)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3992518A (en) * | 1974-10-24 | 1976-11-16 | G. D. Searle & Co. | Method for making a microsealed delivery device |
US4088864A (en) * | 1974-11-18 | 1978-05-09 | Alza Corporation | Process for forming outlet passageways in pills using a laser |
US4200984A (en) * | 1979-03-12 | 1980-05-06 | Fink Ray D | Detachable tool combining bracket and method |
US4284444A (en) * | 1977-08-01 | 1981-08-18 | Herculite Protective Fabrics Corporation | Activated polymer materials and process for making same |
US4398908A (en) * | 1980-11-28 | 1983-08-16 | Siposs George G | Insulin delivery system |
US4435173A (en) * | 1982-03-05 | 1984-03-06 | Delta Medical Industries | Variable rate syringe pump for insulin delivery |
US4542025A (en) * | 1982-07-29 | 1985-09-17 | The Stolle Research And Development Corporation | Injectable, long-acting microparticle formulation for the delivery of anti-inflammatory agents |
US4684479A (en) * | 1985-08-14 | 1987-08-04 | Arrigo Joseph S D | Surfactant mixtures, stable gas-in-liquid emulsions, and methods for the production of such emulsions from said mixtures |
US4861800A (en) * | 1987-08-18 | 1989-08-29 | Buyske Donald A | Method for administering the drug deprenyl so as to minimize the danger of side effects |
US4897268A (en) * | 1987-08-03 | 1990-01-30 | Southern Research Institute | Drug delivery system and method of making the same |
US4933185A (en) * | 1986-09-24 | 1990-06-12 | Massachusetts Institute Of Technology | System for controlled release of biologically active compounds |
US4988337A (en) * | 1988-08-10 | 1991-01-29 | Terumo Kabushiki Kaisha | Syringe pump apparatus |
US5017378A (en) * | 1989-05-01 | 1991-05-21 | The University Of Virginia Alumni Patents Foundation | Intraorgan injection of biologically active compounds contained in slow-release microcapsules or microspheres |
US5089474A (en) * | 1989-05-31 | 1992-02-18 | Roussel Uclaf | Novel microproteins |
US5176502A (en) * | 1990-04-25 | 1993-01-05 | Becton, Dickinson And Company | Syringe pump and the like for delivering medication |
US5186938A (en) * | 1984-07-24 | 1993-02-16 | Key Pharmaceuticals, Inc. | Adhesive transdermal dosage layer |
US5215680A (en) * | 1990-07-10 | 1993-06-01 | Cavitation-Control Technology, Inc. | Method for the production of medical-grade lipid-coated microbubbles, paramagnetic labeling of such microbubbles and therapeutic uses of microbubbles |
US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5298022A (en) * | 1989-05-29 | 1994-03-29 | Amplifon Spa | Wearable artificial pancreas |
US5318540A (en) * | 1990-04-02 | 1994-06-07 | Pharmetrix Corporation | Controlled release infusion device |
US5407609A (en) * | 1989-05-04 | 1995-04-18 | Southern Research Institute | Microencapsulation process and products therefrom |
US5492534A (en) * | 1990-04-02 | 1996-02-20 | Pharmetrix Corporation | Controlled release portable pump |
US5554730A (en) * | 1993-03-09 | 1996-09-10 | Middlesex Sciences, Inc. | Method and kit for making a polysaccharide-protein conjugate |
US5573776A (en) * | 1992-12-02 | 1996-11-12 | Alza Corporation | Oral osmotic device with hydrogel driving member |
US5578709A (en) * | 1993-03-09 | 1996-11-26 | Middlesex Sciences, Inc. | Macromolecular microparticles and methods of production |
US5599907A (en) * | 1989-05-10 | 1997-02-04 | Somatogen, Inc. | Production and use of multimeric hemoglobins |
US5660848A (en) * | 1994-11-02 | 1997-08-26 | The Population Council, Center For Biomedical Research | Subdermally implantable device |
US5874104A (en) * | 1986-08-21 | 1999-02-23 | Nexstar Pharmaceuticals, Inc. | Treatment of systemic fungal infections with phospholipid particles encapsulating polyene antibiotics |
US5916588A (en) * | 1984-04-12 | 1999-06-29 | The Liposome Company, Inc. | Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use |
US5942252A (en) * | 1986-10-24 | 1999-08-24 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissues and controlling their release |
US5965156A (en) * | 1987-11-12 | 1999-10-12 | Nexstar Pharmaceuticals, Inc. | Amphotericin B liposome preparation |
US5981719A (en) * | 1993-03-09 | 1999-11-09 | Epic Therapeutics, Inc. | Macromolecular microparticles and methods of production and use |
US6043094A (en) * | 1996-10-11 | 2000-03-28 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method |
US6056973A (en) * | 1996-10-11 | 2000-05-02 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method of preparation |
US6090925A (en) * | 1993-03-09 | 2000-07-18 | Epic Therapeutics, Inc. | Macromolecular microparticles and methods of production and use |
US6110498A (en) * | 1996-10-25 | 2000-08-29 | Shire Laboratories, Inc. | Osmotic drug delivery system |
US6126966A (en) * | 1996-08-23 | 2000-10-03 | Sequus Pharmaceuticals, Inc. | Liposomes containing a cisplatin compound |
US6183770B1 (en) * | 1999-04-15 | 2001-02-06 | Acutek International | Carrier patch for the delivery of agents to the skin |
US6254573B1 (en) * | 1998-02-05 | 2001-07-03 | Biosense, Inc. | Intracardiac drug delivery device utilizing spring-loaded mechanism |
US6294170B1 (en) * | 1997-08-08 | 2001-09-25 | Amgen Inc. | Composition and method for treating inflammatory diseases |
US6294191B1 (en) * | 1996-10-15 | 2001-09-25 | The Liposome Company, Inc. | N-acyl phosphatidylethanolamine-mediated liposomal drug delivery |
US6294201B1 (en) * | 1997-10-12 | 2001-09-25 | Bayer Aktiengesellschaft | Osmotic medicament releasing system |
US6303148B1 (en) * | 1996-11-19 | 2001-10-16 | Octoplus B.V. | Process for the preparation of a controlled release system |
US6352721B1 (en) * | 2000-01-14 | 2002-03-05 | Osmotica Corp. | Combined diffusion/osmotic pumping drug delivery system |
US6352716B1 (en) * | 1984-04-12 | 2002-03-05 | The Liposome Company Inc. | Steroidal liposomes |
US6361796B1 (en) * | 1996-10-25 | 2002-03-26 | Shire Laboratories, Inc. | Soluble form osmotic dose delivery system |
US20020042079A1 (en) * | 1994-02-01 | 2002-04-11 | Sanford M. Simon | Methods and agents for measuring and controlling multidrug resistance |
US6395302B1 (en) * | 1996-11-19 | 2002-05-28 | Octoplus B.V. | Method for the preparation of microspheres which contain colloidal systems |
US6406713B1 (en) * | 1987-03-05 | 2002-06-18 | The Liposome Company, Inc. | Methods of preparing low-toxicity drug-lipid complexes |
US6458387B1 (en) * | 1999-10-18 | 2002-10-01 | Epic Therapeutics, Inc. | Sustained release microspheres |
US6517859B1 (en) * | 1990-05-16 | 2003-02-11 | Southern Research Institute | Microcapsules for administration of neuroactive agents |
US20030049689A1 (en) * | 2000-12-14 | 2003-03-13 | Cynthia Edwards | Multifunctional polypeptides |
US6534090B2 (en) * | 2001-02-02 | 2003-03-18 | Sun Pharmaceutical Advanced Research Centre Limited | Oral osmotic controlled drug delivery system for a sparingly soluble drug |
US6572585B2 (en) * | 2001-07-12 | 2003-06-03 | Soo Bong Choi | Remote-controlled portable automatic syringe device |
US20030181381A1 (en) * | 1997-02-27 | 2003-09-25 | Michele Himmelspach | Factor X analogues having a modified protease cleavage site |
US20030190740A1 (en) * | 1998-10-13 | 2003-10-09 | The University Of Georgia Research Foundation, Inc | Stabilized bioactive peptides and methods of identification, synthesis, and use |
US20040043446A1 (en) * | 2001-10-19 | 2004-03-04 | Neose Technologies, Inc. | Alpha galalctosidase a: remodeling and glycoconjugation of alpha galactosidase A |
US6713086B2 (en) * | 1998-12-18 | 2004-03-30 | Abbott Laboratories | Controlled release formulation of divalproex sodium |
US6715485B1 (en) * | 1999-03-03 | 2004-04-06 | Optinose As | Nasal delivery device |
US6743211B1 (en) * | 1999-11-23 | 2004-06-01 | Georgia Tech Research Corporation | Devices and methods for enhanced microneedle penetration of biological barriers |
US20040106118A1 (en) * | 2000-10-26 | 2004-06-03 | Harald Kolmar | Method for exposing peptides and polypeptides on the cell surface of bacteria |
US6759057B1 (en) * | 1986-06-12 | 2004-07-06 | The Liposome Company, Inc. | Methods and compositions using liposome-encapsulated non-steroidal anti-inflammatory drugs |
US6838093B2 (en) * | 2001-06-01 | 2005-01-04 | Shire Laboratories, Inc. | System for osmotic delivery of pharmaceutically active agents |
US20050042721A1 (en) * | 2003-06-03 | 2005-02-24 | Jianmin Fang | Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site |
US20050048512A1 (en) * | 2001-04-26 | 2005-03-03 | Avidia Research Institute | Combinatorial libraries of monomer domains |
US6890918B2 (en) * | 2001-04-30 | 2005-05-10 | Shire Laboratories, Inc. | Pharmaceutical compositions including ACE/NEP inhibitors and bioavailability enhancers |
US20050118136A1 (en) * | 2001-03-21 | 2005-06-02 | Cell Therapeutics, Inc. | Recombinant production of polyanionic polymers, and uses thereof |
US20050123997A1 (en) * | 2003-10-30 | 2005-06-09 | Lollar John S. | Modified fVIII having reduced immunogenicity through mutagenesis of A2 and C2 epitopes |
US6905688B2 (en) * | 2000-04-12 | 2005-06-14 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US6945952B2 (en) * | 2002-06-25 | 2005-09-20 | Theraject, Inc. | Solid solution perforator for drug delivery and other applications |
US20060026719A1 (en) * | 2004-01-14 | 2006-02-02 | Kieliszewski Marcia J | Methods of producing peptides/proteins in plants and peptides/proteins produced thereby |
US20060084113A1 (en) * | 1989-09-01 | 2006-04-20 | Ladner Robert C | Directed evolution of disulfide-bonded micro-proteins |
US7045318B2 (en) * | 1995-12-30 | 2006-05-16 | Delta Biotechnology Limited | Recombinant fusion proteins to growth hormone and serum albumin |
US20070048282A1 (en) * | 2004-02-09 | 2007-03-01 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20070161087A1 (en) * | 2003-05-29 | 2007-07-12 | Wolfgang Glaesner | Glp-1 fusion proteins |
US20070191272A1 (en) * | 2005-09-27 | 2007-08-16 | Stemmer Willem P | Proteinaceous pharmaceuticals and uses thereof |
US20070203058A1 (en) * | 2003-09-19 | 2007-08-30 | Novo Nordisk A/S | Novel Glp-1 Derivatives |
US20070244301A1 (en) * | 2006-03-31 | 2007-10-18 | Juergen Siekmann | Pegylated factor VIII |
US20080039341A1 (en) * | 2005-09-27 | 2008-02-14 | Volker Schellenberger | Unstructured recombinant polymers and uses thereof |
US20080167238A1 (en) * | 2001-12-21 | 2008-07-10 | Human Genome Sciences, Inc. | Albumin Fusion Proteins |
US7442778B2 (en) * | 2004-09-24 | 2008-10-28 | Amgen Inc. | Modified Fc molecules |
US20090060862A1 (en) * | 2005-04-06 | 2009-03-05 | Immunomedics, Inc. | PEGylation by the Dock and Lock (DNL) Technique |
US7514257B2 (en) * | 2001-12-07 | 2009-04-07 | Toolgen, Inc. | Zinc finger transcription factor differentiation proteins |
US20090092582A1 (en) * | 2007-08-15 | 2009-04-09 | Oren Bogin | Compositions and methods for modifying properties of biologically active polypeptides |
US20090099031A1 (en) * | 2005-09-27 | 2009-04-16 | Stemmer Willem P | Genetic package and uses thereof |
US7528242B2 (en) * | 1999-11-10 | 2009-05-05 | Rigel Pharmaceuticals, Inc. | Methods and compositions comprising Renilla GFP |
US20100239554A1 (en) * | 2009-02-03 | 2010-09-23 | Amunix Inc. a Delaware Corporation | Extended recombinant polypeptides and compositions comprising same |
US20110151433A1 (en) * | 2005-09-27 | 2011-06-23 | Amunix Operating, Inc. | Methods for production of unstructured recombinant polymers and uses thereof |
Family Cites Families (110)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ZA811368B (en) | 1980-03-24 | 1982-04-28 | Genentech Inc | Bacterial polypedtide expression employing tryptophan promoter-operator |
US4599311A (en) | 1982-08-13 | 1986-07-08 | Kawasaki Glenn H | Glycolytic promotersfor regulated protein expression: protease inhibitor |
US4713339A (en) | 1983-01-19 | 1987-12-15 | Genentech, Inc. | Polycistronic expression vector construction |
US4870008A (en) | 1983-08-12 | 1989-09-26 | Chiron Corporation | Secretory expression in eukaryotes |
US4757006A (en) | 1983-10-28 | 1988-07-12 | Genetics Institute, Inc. | Human factor VIII:C gene and recombinant methods for production |
US5004804A (en) | 1984-01-12 | 1991-04-02 | Nordisk Gentofte | Method and composition for preparation of factor VIIIC |
US4965199A (en) | 1984-04-20 | 1990-10-23 | Genentech, Inc. | Preparation of functional human factor VIII in mammalian cells using methotrexate based selection |
US4931373A (en) | 1984-05-25 | 1990-06-05 | Zymogenetics, Inc. | Stable DNA constructs for expression of α-1 antitrypsin |
US4766073A (en) | 1985-02-25 | 1988-08-23 | Zymogenetics Inc. | Expression of biologically active PDGF analogs in eucaryotic cells |
US4885249A (en) | 1984-12-05 | 1989-12-05 | Allelix, Inc. | Aspergillus niger transformation system |
KR910006424B1 (en) | 1985-08-21 | 1991-08-24 | 인코텍스 비.브이 | Method of manufacturing knitted briefs |
US4882279A (en) | 1985-10-25 | 1989-11-21 | Phillips Petroleum Company | Site selective genomic modification of yeast of the genus pichia |
US5250421A (en) | 1986-01-03 | 1993-10-05 | Genetics Institute, Inc. | Method for producing factor VIII:C-type proteins |
US5198349A (en) | 1986-01-03 | 1993-03-30 | Genetics Institute, Inc. | Method for producing factor VIII:C and analogs |
JP2525022B2 (en) | 1986-01-03 | 1996-08-14 | ジェネティックス・インスチチュ−ト・インコ−ポレ−テッド | VIII: Improved production method for c-factor protein |
US4935349A (en) | 1986-01-17 | 1990-06-19 | Zymogenetics, Inc. | Expression of higher eucaryotic genes in aspergillus |
DK122686D0 (en) | 1986-03-17 | 1986-03-17 | Novo Industri As | PREPARATION OF PROTEINS |
GB8610600D0 (en) | 1986-04-30 | 1986-06-04 | Novo Industri As | Transformation of trichoderma |
US5543502A (en) | 1986-06-24 | 1996-08-06 | Novo Nordisk A/S | Process for producing a coagulation active complex of factor VIII fragments |
DD257197A1 (en) | 1987-01-28 | 1988-06-08 | Univ Dresden Tech | METHOD FOR THE PRODUCTION OF IMMOBILIZED INSULIN PRAEPARATIONS STABILIZED IN ITS BIOLOGICAL ACTIVITY AND MODIFIED IN ITS PHARMACOLOGICAL PROPERTIES |
US6060447A (en) | 1987-05-19 | 2000-05-09 | Chiron Corporation | Protein complexes having Factor VIII:C activity and production thereof |
IL86693A (en) | 1987-06-12 | 1994-06-24 | Stichting Centraal Lab | Proteins with factor VIII activity, process for their preparation using genetically engineered cells and pharmaceutical compositions containing them |
US6346513B1 (en) | 1987-06-12 | 2002-02-12 | Baxter Trading Gmbh | Proteins with factor VIII activity: process for their preparation using genetically-engineered cells and pharmaceutical compositions containing them |
DE3720246A1 (en) | 1987-06-19 | 1988-12-29 | Behringwerke Ag | FACTOR VIII: C-LIKE MOLECULE WITH COAGULATION ACTIVITY |
US4976696A (en) | 1987-08-10 | 1990-12-11 | Becton, Dickinson And Company | Syringe pump and the like for delivering medication |
FR2619314B1 (en) | 1987-08-11 | 1990-06-15 | Transgene Sa | FACTOR VIII ANALOG, PREPARATION METHOD AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME |
US5270176A (en) * | 1987-11-20 | 1993-12-14 | Hoechst Aktiengesellschaft | Method for the selective cleavage of fusion proteins with lysostaphin |
GB8807504D0 (en) | 1988-03-29 | 1988-05-05 | Sandoz Ltd | Improvements in/relating to organic compounds |
NL8900117A (en) * | 1988-05-09 | 1989-12-01 | Hollandse Signaalapparaten Bv | SYSTEM FOR DETERMINING THE ROTATION POSITION OF AN ARTICLE ROTATABLE ON AN AXLE. |
US5037743A (en) | 1988-08-05 | 1991-08-06 | Zymogenetics, Inc. | BAR1 secretion signal |
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 |
US6225449B1 (en) | 1991-10-04 | 2001-05-01 | Washington University | Hormone analogs with multiple CTP extensions |
JP3789928B2 (en) | 1989-10-12 | 2006-06-28 | オハイオ ユニバーシティ | Growth hormone antagonist |
SK60893A3 (en) | 1990-12-13 | 1993-10-06 | Upjohn Co | Fusion polypeptides |
US5206161A (en) | 1991-02-01 | 1993-04-27 | Genentech, Inc. | Human plasma carboxypeptidase B |
US20060122376A1 (en) | 1991-02-07 | 2006-06-08 | Chiron Corporation | Protein complexes having factor VIII:C activity and production thereof |
KR100303872B1 (en) | 1992-10-02 | 2001-11-22 | 크리스터 발스트룀, 프레드릭 베르그, 하랄트 알름 | Compositions containing coagulation factor formulations, methods for their preparation and use of surfactants as stabilizers |
SE504074C2 (en) | 1993-07-05 | 1996-11-04 | Pharmacia Ab | Protein preparation for subcutaneous, intramuscular or intradermal administration |
US5576291A (en) | 1993-09-13 | 1996-11-19 | Baxter International Inc. | Activated factor VIII as a therapeutic agent and method of treating factor VIII deficiency |
US6818439B1 (en) | 1994-12-30 | 2004-11-16 | Chiron Corporation | Methods for administration of recombinant gene delivery vehicles for treatment of hemophilia and other disorders |
US5833991A (en) | 1995-04-10 | 1998-11-10 | Cobra Therapeutics, Ltd. | Glycine-containing sequences conferring invisibility to the immune system |
US5789379A (en) | 1995-04-14 | 1998-08-04 | Allelix Biopharmaceutical Inc. | Glucagon-like peptide-2 analogs |
US5833911A (en) | 1995-05-31 | 1998-11-10 | Johnson & Johnson Medical, Inc. | Method for forming a glove using a folded glove form |
CA2230492C (en) | 1995-09-21 | 2009-05-26 | Genentech, Inc. | Human growth hormone variants |
US7846455B2 (en) | 1996-07-15 | 2010-12-07 | The United States Of America As Represented By The Department Of Health And Human Services | Attenuated chimeric respiratory syncytial virus |
SE9503380D0 (en) | 1995-09-29 | 1995-09-29 | Pharmacia Ab | Protein derivatives |
US6441025B2 (en) | 1996-03-12 | 2002-08-27 | Pg-Txl Company, L.P. | Water soluble paclitaxel derivatives |
PL189698B1 (en) | 1996-03-12 | 2005-09-30 | Pg Txl Co | Water-soluble promedicines of paklitaxel |
US7560107B2 (en) | 1996-06-26 | 2009-07-14 | Emory University | Modified factor VIII |
US6458563B1 (en) | 1996-06-26 | 2002-10-01 | Emory University | Modified factor VIII |
AU5065198A (en) | 1996-11-15 | 1998-06-10 | Maria Grazia Masucci | Fusion proteins having increased half-lives |
CA2225189C (en) | 1997-03-06 | 2010-05-25 | Queen's University At Kingston | Canine factor viii gene, protein and methods of use |
AU736549B2 (en) | 1997-05-21 | 2001-08-02 | Merck Patent Gesellschaft Mit Beschrankter Haftung | Method for the production of non-immunogenic proteins |
US20050260605A1 (en) | 1998-02-11 | 2005-11-24 | Maxygen, Inc. | Targeting of genetic vaccine vectors |
JP2002507393A (en) | 1998-02-11 | 2002-03-12 | マキシジェン, インコーポレイテッド | Antigen library immunization |
US6406632B1 (en) | 1998-04-03 | 2002-06-18 | Symyx Technologies, Inc. | Rapid characterization of polymers |
GB9815157D0 (en) | 1998-07-13 | 1998-09-09 | Metron Designs Ltd | High resolution pulse width setting from relatively low frequency clocks |
WO2000028021A1 (en) | 1998-11-10 | 2000-05-18 | Baxter Aktiengesellschaft | A factor viii-polypeptide with factor viii:c-activity |
US6329186B1 (en) | 1998-12-07 | 2001-12-11 | Novozymes A/S | Glucoamylases with N-terminal extensions |
US20030109428A1 (en) | 1999-12-01 | 2003-06-12 | John Bertin | Novel molecules of the card-related protein family and uses thereof |
US20050287153A1 (en) | 2002-06-28 | 2005-12-29 | Genentech, Inc. | Serum albumin binding peptides for tumor targeting |
EP1355630B1 (en) | 2000-08-15 | 2009-11-25 | The Board Of Trustees Of The University Of Illinois | Method of forming microparticles |
ATE386538T1 (en) | 2001-02-05 | 2008-03-15 | Novo Nordisk Healthcare Ag | COMBINED USE OF FACTOR VII POLYPEPTIDES AND FACTOR VIII POLYPEPTIDES |
WO2002079415A2 (en) | 2001-03-30 | 2002-10-10 | Lexigen Pharmaceuticals Corp. | Reducing the immunogenicity of fusion proteins |
US20040259780A1 (en) | 2001-07-30 | 2004-12-23 | Glasebrook Andrew Lawrence | Method for treating diabetes and obesity |
US7081446B2 (en) | 2002-01-31 | 2006-07-25 | The Trustees Of Columbia University In The City Of New York | Long-acting follicle stimulating hormone analogues and uses thereof |
US7294513B2 (en) | 2002-07-24 | 2007-11-13 | Wyatt Technology Corporation | Method and apparatus for characterizing solutions of small particles |
US20040142870A1 (en) | 2002-11-20 | 2004-07-22 | Finn Rory F. | N-terminally monopegylated human growth hormone conjugates, process for their preparation, and methods of use thereof |
US20050032081A1 (en) | 2002-12-13 | 2005-02-10 | Jingyue Ju | Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry |
US20050176108A1 (en) | 2003-03-13 | 2005-08-11 | Young-Min Kim | Physiologically active polypeptide conjugate having prolonged in vivo half-life |
TW200502222A (en) | 2003-04-02 | 2005-01-16 | Novartis Ag | Use of 10-hydroxy-10,11-dihydrocarbamazepine derivatives for the treatment of affective disorders |
EP1633382A4 (en) | 2003-05-14 | 2009-08-05 | Dow Corning | Repeat sequence protein polymer active agent conjugates, methods and uses |
US20050152979A1 (en) | 2003-09-05 | 2005-07-14 | Cell Therapeutics, Inc. | Hydrophobic drug compositions containing reconstitution enhancer |
US20060040856A1 (en) | 2003-12-03 | 2006-02-23 | Neose Technologies, Inc. | Glycopegylated factor IX |
US20050143303A1 (en) | 2003-12-26 | 2005-06-30 | Nastech Pharmaceutical Company Inc. | Intranasal administration of glucose-regulating peptides |
US8076288B2 (en) | 2004-02-11 | 2011-12-13 | Amylin Pharmaceuticals, Inc. | Hybrid polypeptides having glucose lowering activity |
EP2332977B1 (en) * | 2004-07-23 | 2015-11-25 | Acceleron Pharma Inc. | ActRII receptor polypeptides |
EA200700380A1 (en) | 2004-08-31 | 2007-10-26 | Фармация Энд Апджон Компани Ллс | CONJUGATES OF GLYCEROLICOLORBED POLYETHYLENE GLYCOL - HUMAN GROWTH HORMONE, METHOD OF THEIR PRODUCTION AND METHODS OF THEIR APPLICATION |
KR101654011B1 (en) | 2004-11-12 | 2016-09-05 | 바이엘 헬스케어 엘엘씨 | Site-directed modification of fviii |
MX2007008982A (en) | 2005-01-25 | 2007-12-06 | Cell Therapeutics Inc | Conjugates of biologically active proteins having a modified in vivo half-life. |
US8263545B2 (en) | 2005-02-11 | 2012-09-11 | Amylin Pharmaceuticals, Inc. | GIP analog and hybrid polypeptides with selectable properties |
EP1871801A2 (en) | 2005-04-01 | 2008-01-02 | Novo Nordisk Health Care AG | Blood coagulation fviii analogues |
DE102005038063A1 (en) * | 2005-08-10 | 2007-02-15 | Schneider Gmbh + Co. Kg | Preformed block piece with three support points |
JP2009525946A (en) | 2005-12-20 | 2009-07-16 | デューク・ユニヴァーシティ | Methods and compositions for delivering active agents with enhanced pharmacological properties |
EP1996937A4 (en) | 2006-03-06 | 2009-04-08 | Amunix Inc | Genetic packages and uses thereof |
WO2007130535A2 (en) | 2006-05-04 | 2007-11-15 | Molecular Innovations | Novel protein fusion/tag technology |
EP2446904B1 (en) | 2006-05-30 | 2015-04-29 | Genentech, Inc. | Anti-CD22 antibodies, their immunoconjugates and uses thereof |
WO2008012629A2 (en) | 2006-07-24 | 2008-01-31 | Biorexis Pharmaceutical Corporation | Exendin fusion proteins |
ES2636089T3 (en) | 2006-10-27 | 2017-10-05 | Genentech, Inc. | Antibodies and immunoconjugates and uses for them |
WO2008049711A1 (en) | 2006-10-27 | 2008-05-02 | Novo Nordisk A/S | Peptide extended insulins |
CN101190945A (en) | 2006-11-29 | 2008-06-04 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation for specificity anticoagulant substance and application thereof |
DE602008005596D1 (en) | 2007-06-21 | 2011-04-28 | Univ Muenchen Tech | BIOLOGICALLY ACTIVE PROTEINS WITH INCREASED IN VIVO AND / OR IN VITRO STABILITY |
EP2307038A4 (en) | 2008-06-27 | 2013-03-27 | Univ Duke | Therapeutic agents comprising elastin-like peptides |
US8680050B2 (en) | 2009-02-03 | 2014-03-25 | Amunix Operating Inc. | Growth hormone polypeptides fused to extended recombinant polypeptides and methods of making and using same |
US8703717B2 (en) | 2009-02-03 | 2014-04-22 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
EA030619B1 (en) | 2009-06-08 | 2018-09-28 | Амуникс Оперейтинг Инк. | Fusion protein comprising growth hormone sequences and xten, method of producing and using the same |
US9849188B2 (en) | 2009-06-08 | 2017-12-26 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
AU2010258898B8 (en) | 2009-06-08 | 2015-02-05 | Amunix Operating Inc. | Glucose-regulating polypeptides and methods of making and using same |
US20120263701A1 (en) | 2009-08-24 | 2012-10-18 | Volker Schellenberger | Coagulation factor vii compositions and methods of making and using same |
HUE036233T2 (en) | 2009-12-06 | 2018-06-28 | Bioverativ Therapeutics Inc | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof |
WO2011084808A2 (en) | 2009-12-21 | 2011-07-14 | Amunix Operating Inc. | Bifunctional polypeptide compositions and methods for treatment of metabolic and cardiovascular diseases |
EP2552967A4 (en) | 2010-04-02 | 2014-10-08 | Amunix Operating Inc | Binding fusion proteins, binding fusion protein-drug conjugates, xten-drug conjugates and methods of making and using same |
WO2011123830A2 (en) | 2010-04-02 | 2011-10-06 | Amunix Operating Inc. | Alpha 1-antitrypsin compositions and methods of making and using same |
EP3604264A1 (en) | 2010-04-27 | 2020-02-05 | SynAffix B.V. | Fused cyclooctyne compounds |
EP3508573A1 (en) | 2010-07-09 | 2019-07-10 | Bioverativ Therapeutics Inc. | Systems for factor viii processing and methods thereof |
UA115120C2 (en) | 2010-07-09 | 2017-09-25 | Байоджен Хемофіліа Інк. | Factor ix polypeptides and methods fo use thereof |
KR20140002601A (en) | 2010-07-09 | 2014-01-08 | 바이오겐 이데크 헤모필리아 인코포레이티드 | Chimeric clotting factors |
JP2013532176A (en) | 2010-07-15 | 2013-08-15 | ノヴォ ノルディスク アー/エス | Stabilized factor VIII variant |
US20130017997A1 (en) | 2010-08-19 | 2013-01-17 | Amunix Operating Inc. | Factor VIII Compositions and Methods of Making and Using Same |
SG11201408152VA (en) | 2012-06-05 | 2015-01-29 | Amunix Operating Inc | Hgh-xten fusion protein and its use in the treatment of growth hormone deficiency |
-
2007
- 2007-03-06 US US11/715,276 patent/US7855279B2/en active Active
-
2009
- 2009-12-09 US US12/634,556 patent/US20100189682A1/en not_active Abandoned
-
2010
- 2010-11-03 US US12/939,120 patent/US8492530B2/en active Active
-
2012
- 2012-02-14 US US13/396,403 patent/US20120220011A1/en not_active Abandoned
- 2012-04-04 US US13/439,738 patent/US20120230947A1/en not_active Abandoned
-
2013
- 2013-12-10 US US14/102,297 patent/US20140356326A1/en not_active Abandoned
-
2015
- 2015-12-16 US US14/970,723 patent/US9938331B2/en active Active
Patent Citations (99)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3992518A (en) * | 1974-10-24 | 1976-11-16 | G. D. Searle & Co. | Method for making a microsealed delivery device |
US4088864A (en) * | 1974-11-18 | 1978-05-09 | Alza Corporation | Process for forming outlet passageways in pills using a laser |
US4284444A (en) * | 1977-08-01 | 1981-08-18 | Herculite Protective Fabrics Corporation | Activated polymer materials and process for making same |
US4200984A (en) * | 1979-03-12 | 1980-05-06 | Fink Ray D | Detachable tool combining bracket and method |
US4398908A (en) * | 1980-11-28 | 1983-08-16 | Siposs George G | Insulin delivery system |
US4435173A (en) * | 1982-03-05 | 1984-03-06 | Delta Medical Industries | Variable rate syringe pump for insulin delivery |
US4542025A (en) * | 1982-07-29 | 1985-09-17 | The Stolle Research And Development Corporation | Injectable, long-acting microparticle formulation for the delivery of anti-inflammatory agents |
US5916588A (en) * | 1984-04-12 | 1999-06-29 | The Liposome Company, Inc. | Peptide-containing liposomes, immunogenic liposomes and methods of preparation and use |
US6352716B1 (en) * | 1984-04-12 | 2002-03-05 | The Liposome Company Inc. | Steroidal liposomes |
US5186938A (en) * | 1984-07-24 | 1993-02-16 | Key Pharmaceuticals, Inc. | Adhesive transdermal dosage layer |
US4684479A (en) * | 1985-08-14 | 1987-08-04 | Arrigo Joseph S D | Surfactant mixtures, stable gas-in-liquid emulsions, and methods for the production of such emulsions from said mixtures |
US6759057B1 (en) * | 1986-06-12 | 2004-07-06 | The Liposome Company, Inc. | Methods and compositions using liposome-encapsulated non-steroidal anti-inflammatory drugs |
US5874104A (en) * | 1986-08-21 | 1999-02-23 | Nexstar Pharmaceuticals, Inc. | Treatment of systemic fungal infections with phospholipid particles encapsulating polyene antibiotics |
US4933185A (en) * | 1986-09-24 | 1990-06-12 | Massachusetts Institute Of Technology | System for controlled release of biologically active compounds |
US6024983A (en) * | 1986-10-24 | 2000-02-15 | Southern Research Institute | Composition for delivering bioactive agents for immune response and its preparation |
US5942252A (en) * | 1986-10-24 | 1999-08-24 | Southern Research Institute | Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissues and controlling their release |
US6406713B1 (en) * | 1987-03-05 | 2002-06-18 | The Liposome Company, Inc. | Methods of preparing low-toxicity drug-lipid complexes |
US4897268A (en) * | 1987-08-03 | 1990-01-30 | Southern Research Institute | Drug delivery system and method of making the same |
US4861800A (en) * | 1987-08-18 | 1989-08-29 | Buyske Donald A | Method for administering the drug deprenyl so as to minimize the danger of side effects |
US5965156A (en) * | 1987-11-12 | 1999-10-12 | Nexstar Pharmaceuticals, Inc. | Amphotericin B liposome preparation |
US4988337A (en) * | 1988-08-10 | 1991-01-29 | Terumo Kabushiki Kaisha | Syringe pump apparatus |
US5223409A (en) * | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US20020150881A1 (en) * | 1988-09-02 | 2002-10-17 | Ladner Robert Charles | Directed evolution of novel binding proteins |
US5017378A (en) * | 1989-05-01 | 1991-05-21 | The University Of Virginia Alumni Patents Foundation | Intraorgan injection of biologically active compounds contained in slow-release microcapsules or microspheres |
US5407609A (en) * | 1989-05-04 | 1995-04-18 | Southern Research Institute | Microencapsulation process and products therefrom |
US5599907A (en) * | 1989-05-10 | 1997-02-04 | Somatogen, Inc. | Production and use of multimeric hemoglobins |
US5298022A (en) * | 1989-05-29 | 1994-03-29 | Amplifon Spa | Wearable artificial pancreas |
US5089474A (en) * | 1989-05-31 | 1992-02-18 | Roussel Uclaf | Novel microproteins |
US20060084113A1 (en) * | 1989-09-01 | 2006-04-20 | Ladner Robert C | Directed evolution of disulfide-bonded micro-proteins |
US7413537B2 (en) * | 1989-09-01 | 2008-08-19 | Dyax Corp. | Directed evolution of disulfide-bonded micro-proteins |
US5492534A (en) * | 1990-04-02 | 1996-02-20 | Pharmetrix Corporation | Controlled release portable pump |
US5318540A (en) * | 1990-04-02 | 1994-06-07 | Pharmetrix Corporation | Controlled release infusion device |
US5176502A (en) * | 1990-04-25 | 1993-01-05 | Becton, Dickinson And Company | Syringe pump and the like for delivering medication |
US6517859B1 (en) * | 1990-05-16 | 2003-02-11 | Southern Research Institute | Microcapsules for administration of neuroactive agents |
US5215680A (en) * | 1990-07-10 | 1993-06-01 | Cavitation-Control Technology, Inc. | Method for the production of medical-grade lipid-coated microbubbles, paramagnetic labeling of such microbubbles and therapeutic uses of microbubbles |
US5573776A (en) * | 1992-12-02 | 1996-11-12 | Alza Corporation | Oral osmotic device with hydrogel driving member |
US6090925A (en) * | 1993-03-09 | 2000-07-18 | Epic Therapeutics, Inc. | Macromolecular microparticles and methods of production and use |
US5554730A (en) * | 1993-03-09 | 1996-09-10 | Middlesex Sciences, Inc. | Method and kit for making a polysaccharide-protein conjugate |
US6268053B1 (en) * | 1993-03-09 | 2001-07-31 | Epic Therapeutics, Inc. | Macromolecular microparticles and methods of production and use |
US5981719A (en) * | 1993-03-09 | 1999-11-09 | Epic Therapeutics, Inc. | Macromolecular microparticles and methods of production and use |
US5578709A (en) * | 1993-03-09 | 1996-11-26 | Middlesex Sciences, Inc. | Macromolecular microparticles and methods of production |
US20020042079A1 (en) * | 1994-02-01 | 2002-04-11 | Sanford M. Simon | Methods and agents for measuring and controlling multidrug resistance |
US5756115A (en) * | 1994-11-02 | 1998-05-26 | The Population Coucil, Center For Biomedical Research | Contraceptive method using a subdermally implantable device |
US5660848A (en) * | 1994-11-02 | 1997-08-26 | The Population Council, Center For Biomedical Research | Subdermally implantable device |
US7045318B2 (en) * | 1995-12-30 | 2006-05-16 | Delta Biotechnology Limited | Recombinant fusion proteins to growth hormone and serum albumin |
US6126966A (en) * | 1996-08-23 | 2000-10-03 | Sequus Pharmaceuticals, Inc. | Liposomes containing a cisplatin compound |
US6056973A (en) * | 1996-10-11 | 2000-05-02 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method of preparation |
US6043094A (en) * | 1996-10-11 | 2000-03-28 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method |
US6294191B1 (en) * | 1996-10-15 | 2001-09-25 | The Liposome Company, Inc. | N-acyl phosphatidylethanolamine-mediated liposomal drug delivery |
US6361796B1 (en) * | 1996-10-25 | 2002-03-26 | Shire Laboratories, Inc. | Soluble form osmotic dose delivery system |
US6284276B1 (en) * | 1996-10-25 | 2001-09-04 | Shire Laboratories, Inc. | Soluble form osmotic dose delivery system |
US6110498A (en) * | 1996-10-25 | 2000-08-29 | Shire Laboratories, Inc. | Osmotic drug delivery system |
US6514532B2 (en) * | 1996-10-25 | 2003-02-04 | Shire Laboratories, Inc. | Soluble form osmotic dose delivery system |
US6303148B1 (en) * | 1996-11-19 | 2001-10-16 | Octoplus B.V. | Process for the preparation of a controlled release system |
US6395302B1 (en) * | 1996-11-19 | 2002-05-28 | Octoplus B.V. | Method for the preparation of microspheres which contain colloidal systems |
US6733753B2 (en) * | 1997-02-10 | 2004-05-11 | Amgen Inc. | Composition and method for treating inflammatory diseases |
US20030181381A1 (en) * | 1997-02-27 | 2003-09-25 | Michele Himmelspach | Factor X analogues having a modified protease cleavage site |
US6294170B1 (en) * | 1997-08-08 | 2001-09-25 | Amgen Inc. | Composition and method for treating inflammatory diseases |
US6294201B1 (en) * | 1997-10-12 | 2001-09-25 | Bayer Aktiengesellschaft | Osmotic medicament releasing system |
US6309370B1 (en) * | 1998-02-05 | 2001-10-30 | Biosense, Inc. | Intracardiac drug delivery |
US6254573B1 (en) * | 1998-02-05 | 2001-07-03 | Biosense, Inc. | Intracardiac drug delivery device utilizing spring-loaded mechanism |
US20030190740A1 (en) * | 1998-10-13 | 2003-10-09 | The University Of Georgia Research Foundation, Inc | Stabilized bioactive peptides and methods of identification, synthesis, and use |
US6713086B2 (en) * | 1998-12-18 | 2004-03-30 | Abbott Laboratories | Controlled release formulation of divalproex sodium |
US6715485B1 (en) * | 1999-03-03 | 2004-04-06 | Optinose As | Nasal delivery device |
US6183770B1 (en) * | 1999-04-15 | 2001-02-06 | Acutek International | Carrier patch for the delivery of agents to the skin |
US6458387B1 (en) * | 1999-10-18 | 2002-10-01 | Epic Therapeutics, Inc. | Sustained release microspheres |
US7528242B2 (en) * | 1999-11-10 | 2009-05-05 | Rigel Pharmaceuticals, Inc. | Methods and compositions comprising Renilla GFP |
US6743211B1 (en) * | 1999-11-23 | 2004-06-01 | Georgia Tech Research Corporation | Devices and methods for enhanced microneedle penetration of biological barriers |
US6352721B1 (en) * | 2000-01-14 | 2002-03-05 | Osmotica Corp. | Combined diffusion/osmotic pumping drug delivery system |
US6905688B2 (en) * | 2000-04-12 | 2005-06-14 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US20040106118A1 (en) * | 2000-10-26 | 2004-06-03 | Harald Kolmar | Method for exposing peptides and polypeptides on the cell surface of bacteria |
US20030049689A1 (en) * | 2000-12-14 | 2003-03-13 | Cynthia Edwards | Multifunctional polypeptides |
US6534090B2 (en) * | 2001-02-02 | 2003-03-18 | Sun Pharmaceutical Advanced Research Centre Limited | Oral osmotic controlled drug delivery system for a sparingly soluble drug |
US20080176288A1 (en) * | 2001-03-21 | 2008-07-24 | Cell Therapeutics, Inc. | Recombinant Production of Polyanionic Polymers, and Uses Thereof |
US20050118136A1 (en) * | 2001-03-21 | 2005-06-02 | Cell Therapeutics, Inc. | Recombinant production of polyanionic polymers, and uses thereof |
US20050048512A1 (en) * | 2001-04-26 | 2005-03-03 | Avidia Research Institute | Combinatorial libraries of monomer domains |
US6890918B2 (en) * | 2001-04-30 | 2005-05-10 | Shire Laboratories, Inc. | Pharmaceutical compositions including ACE/NEP inhibitors and bioavailability enhancers |
US6838093B2 (en) * | 2001-06-01 | 2005-01-04 | Shire Laboratories, Inc. | System for osmotic delivery of pharmaceutically active agents |
US6572585B2 (en) * | 2001-07-12 | 2003-06-03 | Soo Bong Choi | Remote-controlled portable automatic syringe device |
US20040043446A1 (en) * | 2001-10-19 | 2004-03-04 | Neose Technologies, Inc. | Alpha galalctosidase a: remodeling and glycoconjugation of alpha galactosidase A |
US7514257B2 (en) * | 2001-12-07 | 2009-04-07 | Toolgen, Inc. | Zinc finger transcription factor differentiation proteins |
US20080167238A1 (en) * | 2001-12-21 | 2008-07-10 | Human Genome Sciences, Inc. | Albumin Fusion Proteins |
US6945952B2 (en) * | 2002-06-25 | 2005-09-20 | Theraject, Inc. | Solid solution perforator for drug delivery and other applications |
US20070161087A1 (en) * | 2003-05-29 | 2007-07-12 | Wolfgang Glaesner | Glp-1 fusion proteins |
US20050042721A1 (en) * | 2003-06-03 | 2005-02-24 | Jianmin Fang | Compositions and methods for enhanced expression of recombinant polypeptides from a single vector using a peptide cleavage site |
US20070203058A1 (en) * | 2003-09-19 | 2007-08-30 | Novo Nordisk A/S | Novel Glp-1 Derivatives |
US20050123997A1 (en) * | 2003-10-30 | 2005-06-09 | Lollar John S. | Modified fVIII having reduced immunogenicity through mutagenesis of A2 and C2 epitopes |
US20060026719A1 (en) * | 2004-01-14 | 2006-02-02 | Kieliszewski Marcia J | Methods of producing peptides/proteins in plants and peptides/proteins produced thereby |
US20070048282A1 (en) * | 2004-02-09 | 2007-03-01 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US7442778B2 (en) * | 2004-09-24 | 2008-10-28 | Amgen Inc. | Modified Fc molecules |
US20090060862A1 (en) * | 2005-04-06 | 2009-03-05 | Immunomedics, Inc. | PEGylation by the Dock and Lock (DNL) Technique |
US20080039341A1 (en) * | 2005-09-27 | 2008-02-14 | Volker Schellenberger | Unstructured recombinant polymers and uses thereof |
US20070212703A1 (en) * | 2005-09-27 | 2007-09-13 | Stemmer Willem P | Proteinaceous pharmaceuticals and uses thereof |
US20070191272A1 (en) * | 2005-09-27 | 2007-08-16 | Stemmer Willem P | Proteinaceous pharmaceuticals and uses thereof |
US20090099031A1 (en) * | 2005-09-27 | 2009-04-16 | Stemmer Willem P | Genetic package and uses thereof |
US20110151433A1 (en) * | 2005-09-27 | 2011-06-23 | Amunix Operating, Inc. | Methods for production of unstructured recombinant polymers and uses thereof |
US20070244301A1 (en) * | 2006-03-31 | 2007-10-18 | Juergen Siekmann | Pegylated factor VIII |
US20090092582A1 (en) * | 2007-08-15 | 2009-04-09 | Oren Bogin | Compositions and methods for modifying properties of biologically active polypeptides |
US20100239554A1 (en) * | 2009-02-03 | 2010-09-23 | Amunix Inc. a Delaware Corporation | Extended recombinant polypeptides and compositions comprising same |
Cited By (95)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110171687A1 (en) * | 2005-09-27 | 2011-07-14 | Amunix Operating, Inc. | Unstructured recombinant polymers and compositions comprising same |
US20090099031A1 (en) * | 2005-09-27 | 2009-04-16 | Stemmer Willem P | Genetic package and uses thereof |
US7846445B2 (en) | 2005-09-27 | 2010-12-07 | Amunix Operating, Inc. | Methods for production of unstructured recombinant polymers and uses thereof |
US9938331B2 (en) | 2005-09-27 | 2018-04-10 | Amunix Operating Inc. | Biologically active proteins having increased in vivo and/or in vitro stability |
US20080286808A1 (en) * | 2005-09-27 | 2008-11-20 | Volker Schellenberger | Methods for production of unstructured recombinant polymers and uses thereof |
US8492530B2 (en) | 2005-09-27 | 2013-07-23 | Amunix Operating Inc. | Unstructured recombinant polymers and compositions comprising same |
US20110151433A1 (en) * | 2005-09-27 | 2011-06-23 | Amunix Operating, Inc. | Methods for production of unstructured recombinant polymers and uses thereof |
US20100268055A1 (en) * | 2007-07-19 | 2010-10-21 | Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University | Self-Anchoring MEMS Intrafascicular Neural Electrode |
US20090092582A1 (en) * | 2007-08-15 | 2009-04-09 | Oren Bogin | Compositions and methods for modifying properties of biologically active polypeptides |
US20100260706A1 (en) * | 2007-08-15 | 2010-10-14 | Oren Bogin | Compositions and methods for improving production of recombinant polypeptides |
US8933197B2 (en) | 2007-08-15 | 2015-01-13 | Amunix Operating Inc. | Compositions comprising modified biologically active polypeptides |
US9926351B2 (en) | 2009-02-03 | 2018-03-27 | Amunix Operating Inc. | Extended recombinant polypeptides and compositions comprising same |
US8703717B2 (en) | 2009-02-03 | 2014-04-22 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
US10961287B2 (en) | 2009-02-03 | 2021-03-30 | Amunix Pharmaceuticals, Inc | Extended recombinant polypeptides and compositions comprising same |
US20110172146A1 (en) * | 2009-02-03 | 2011-07-14 | Amunix Operating, Inc. | Growth hormone polypeptides and methods of making and using same |
US9168312B2 (en) | 2009-02-03 | 2015-10-27 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
US20110077199A1 (en) * | 2009-02-03 | 2011-03-31 | Amunix, Inc. | Growth hormone polypeptides and methods of making and using same |
US20100323956A1 (en) * | 2009-02-03 | 2010-12-23 | Amunix, Inc. | Glucose-regulating polypeptides and methods of making and using same |
US8673860B2 (en) | 2009-02-03 | 2014-03-18 | Amunix Operating Inc. | Extended recombinant polypeptides and compositions comprising same |
US8680050B2 (en) | 2009-02-03 | 2014-03-25 | Amunix Operating Inc. | Growth hormone polypeptides fused to extended recombinant polypeptides and methods of making and using same |
US9371369B2 (en) | 2009-02-03 | 2016-06-21 | Amunix Operating Inc. | Extended recombinant polypeptides and compositions comprising same |
US8716448B2 (en) | 2009-02-03 | 2014-05-06 | Amunix Operating Inc. | Coagulation factor VII compositions and methods of making and using same |
US20110046061A1 (en) * | 2009-02-03 | 2011-02-24 | Amunix Operating, Inc. | Coagulation factor VII compositions and methods of making and using same |
US8957021B2 (en) | 2009-02-03 | 2015-02-17 | Amunix Operating Inc. | Glucose-regulating polypeptides and methods of making and using same |
US9849188B2 (en) | 2009-06-08 | 2017-12-26 | Amunix Operating Inc. | Growth hormone polypeptides and methods of making and using same |
US10000543B2 (en) | 2009-06-08 | 2018-06-19 | Amunix Operating Inc. | Glucose-regulating polypeptides and methods of making and using same |
US9540430B2 (en) | 2009-06-08 | 2017-01-10 | Amunix Operating Inc. | Glucose-regulating polypeptides and methods of making and using same |
US20110046060A1 (en) * | 2009-08-24 | 2011-02-24 | Amunix Operating, Inc., | Coagulation factor IX compositions and methods of making and using same |
US9062299B2 (en) | 2009-08-24 | 2015-06-23 | Amunix Operating Inc. | Coagulation factor IX compositions and methods of making and using same |
US9758776B2 (en) | 2009-08-24 | 2017-09-12 | Amunix Operating Inc. | Coagulation factor IX compositions and methods of making and using same |
US9376672B2 (en) | 2009-08-24 | 2016-06-28 | Amunix Operating Inc. | Coagulation factor IX compositions and methods of making and using same |
US11266720B2 (en) | 2009-12-06 | 2022-03-08 | Bioverativ Therapeutics Inc. | Factor VIII-FC chimeric and hybrid polypeptides, and methods of use thereof |
US9241978B2 (en) | 2009-12-06 | 2016-01-26 | Biogen Hemophilia Inc. | Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof |
EP3906937A1 (en) | 2009-12-06 | 2021-11-10 | Bioverativ Therapeutics Inc. | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof |
EP3326643A1 (en) | 2009-12-06 | 2018-05-30 | Bioverativ Therapeutics Inc. | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof |
US9050318B2 (en) | 2009-12-06 | 2015-06-09 | Biogen Idec Hemophilia Inc. | Factor VIII-Fc chimeric and hybrid polypeptides, and methods of use thereof |
EP4382170A2 (en) | 2009-12-06 | 2024-06-12 | Bioverativ Therapeutics Inc. | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof |
US10870874B2 (en) | 2010-04-02 | 2020-12-22 | Amunix Pharmaceuticals, Inc. | Binding fusion proteins, binding fusion protein-drug conjugates, XTEN-drug conjugates and methods of making and using same |
US9976166B2 (en) | 2010-04-02 | 2018-05-22 | Amunix Operating Inc. | Binding fusion proteins, binding fusion protein-drug conjugates, XTEN-drug conjugates and methods of making and using same |
US8557961B2 (en) | 2010-04-02 | 2013-10-15 | Amunix Operating Inc. | Alpha 1-antitrypsin compositions and methods of making and using same |
US9249211B2 (en) | 2010-04-02 | 2016-02-02 | Amunix Operating Inc. | Binding fusion proteins, binding fusion protein-drug conjugates, XTEN-drug conjugates and methods of making and using same |
US9867873B2 (en) | 2010-07-09 | 2018-01-16 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US10561714B2 (en) | 2010-07-09 | 2020-02-18 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
EP3552619A1 (en) | 2010-07-09 | 2019-10-16 | Bioverativ Therapeutics Inc. | Factor ix polypeptides and methods of use thereof |
US9675676B2 (en) | 2010-07-09 | 2017-06-13 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US9670475B2 (en) | 2010-07-09 | 2017-06-06 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US9629903B2 (en) | 2010-07-09 | 2017-04-25 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US9623091B2 (en) | 2010-07-09 | 2017-04-18 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US9233145B2 (en) | 2010-07-09 | 2016-01-12 | Biogen Hemophila Inc. | Factor IX polypeptides and methods of use thereof |
US10898554B1 (en) | 2010-07-09 | 2021-01-26 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US10568943B2 (en) | 2010-07-09 | 2020-02-25 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US10548954B2 (en) | 2010-07-09 | 2020-02-04 | Bioverativ Therapeutics Inc. | Factor IX polypeptides and methods of use thereof |
US20120207743A1 (en) * | 2011-02-14 | 2012-08-16 | Allergan, Inc. | Inhibiting Aberrant Blood Vessel Formation Using Retargeted Endopeptidases |
WO2012142142A3 (en) * | 2011-04-12 | 2012-12-13 | Temple University - Of The Commonwealth System Higher Education | Adiponectin receptor agonists and methods of use |
WO2012142142A2 (en) * | 2011-04-12 | 2012-10-18 | Temple University - Of The Commonwealth System Higher Education | Adiponectin receptor agonists and methods of use |
US9073965B2 (en) | 2011-04-12 | 2015-07-07 | Temple University—Of the Commonwealth System of Higher Education | Adiponectin receptor agonists and methods of use |
EP3527218A1 (en) | 2011-06-10 | 2019-08-21 | Bioverativ Therapeutics Inc. | Pro-coagulant compounds and methods of use thereof |
US9486507B2 (en) | 2011-06-10 | 2016-11-08 | Biogen Ma Inc. | Pro-coagulant compounds and methods of use thereof |
EP3513804A1 (en) | 2011-07-08 | 2019-07-24 | Bioverativ Therapeutics Inc. | Factor viii chimeric and hybrid polypeptides, and methods of use thereof |
EP4169525A1 (en) | 2011-07-08 | 2023-04-26 | Bioverativ Therapeutics Inc. | Factor viii chimeric and hybrid polypeptides, and methods of use thereof |
US10010622B2 (en) | 2011-07-08 | 2018-07-03 | Bioverativ Therapeutics Inc. | Factor VIII chimeric and hybrid polypeptides, and methods of use thereof |
US10881742B2 (en) | 2011-07-08 | 2021-01-05 | Bioverativ Therapeutics Inc. | Factor VIII chimeric and hybrid polypeptides, and methods of use thereof |
US10656167B2 (en) | 2011-07-25 | 2020-05-19 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
US11747351B2 (en) | 2011-07-25 | 2023-09-05 | Bioverativ Therapeutics Inc. | Assays to monitor bleeding disorders |
US11370827B2 (en) * | 2012-01-12 | 2022-06-28 | Bioverativ Therapeutics Inc. | Chimeric factor VIII polypeptides and uses thereof |
US10370430B2 (en) | 2012-02-15 | 2019-08-06 | Bioverativ Therapeutics Inc. | Recombinant factor VIII proteins |
US10421798B2 (en) | 2012-02-15 | 2019-09-24 | Bioverativ Therapeutics Inc. | Factor VIII compositions and methods of making and using same |
US11685771B2 (en) | 2012-02-15 | 2023-06-27 | Bioverativ Therapeutics Inc. | Recombinant factor VIII proteins |
US10953073B2 (en) | 2012-02-27 | 2021-03-23 | Amunix Pharmaceuticals, Inc. | XTEN conjugate compositions and methods of making same |
US10172953B2 (en) | 2012-02-27 | 2019-01-08 | Amunix Operating Inc. | XTEN conjugate compositions and methods of making same |
EP3404105A1 (en) | 2012-07-06 | 2018-11-21 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor viii polypeptides and uses thereof |
US10023628B2 (en) | 2012-07-06 | 2018-07-17 | Bioverativ Therapeutics Inc. | Cell line expressing single chain factor VIII polypeptides and uses thereof |
US11225650B2 (en) | 2012-09-25 | 2022-01-18 | Bioverativ Therapeutics Inc. | Methods of using FIX polypeptides |
US10588949B2 (en) | 2013-03-15 | 2020-03-17 | Bioverativ Therapeutics Inc. | Factor IX polypeptide formulations |
US11642398B2 (en) | 2013-03-15 | 2023-05-09 | Bioverativ Therapeutics Inc. | Factor IX polypeptide formulations |
US10548953B2 (en) | 2013-08-14 | 2020-02-04 | Bioverativ Therapeutics Inc. | Factor VIII-XTEN fusions and uses thereof |
US10584147B2 (en) | 2013-11-08 | 2020-03-10 | Biovertiv Therapeutics Inc. | Procoagulant fusion compound |
WO2015195531A2 (en) | 2014-06-18 | 2015-12-23 | Albert Einstein College Of Medicine, Inc. | Syntac polypeptides and uses thereof |
EP3483179A1 (en) | 2014-06-18 | 2019-05-15 | Albert Einstein College of Medicine, Inc. | Syntac polypeptides and uses thereof |
EP3480213A1 (en) | 2014-06-18 | 2019-05-08 | Albert Einstein College of Medicine, Inc. | Syntac polypeptides and uses thereof |
US10745680B2 (en) | 2015-08-03 | 2020-08-18 | Bioverativ Therapeutics Inc. | Factor IX fusion proteins and methods of making and using same |
US11713358B2 (en) | 2015-08-28 | 2023-08-01 | Amunix Pharmaceuticals, Inc. | Chimeric polypeptide assembly and methods of making and using the same |
US11981744B2 (en) | 2015-08-28 | 2024-05-14 | Amunix Pharmaceuticals, Inc. | Chimeric polypeptide assembly and methods of making and using the same |
US10988522B2 (en) | 2016-02-02 | 2021-04-27 | University Of Southern California | Proteolically resistant cyclotides with angiotensin 1-7 like activity |
WO2017201210A1 (en) | 2016-05-18 | 2017-11-23 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
WO2017201131A1 (en) | 2016-05-18 | 2017-11-23 | Albert Einstein College Of Medicine, Inc. | Variant pd-l1 polypeptides, t-cell modulatory multimeric polypeptides, and methods of use thereof |
EP4360647A2 (en) | 2016-12-22 | 2024-05-01 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
WO2018129474A1 (en) | 2017-01-09 | 2018-07-12 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
WO2019051127A1 (en) | 2017-09-07 | 2019-03-14 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptide with conjugation sites and methods of use thereof |
WO2019139896A1 (en) | 2018-01-09 | 2019-07-18 | Cue Biopharma, Inc. | Multimeric t-cell modulatory polypeptides and methods of use thereof |
US12030925B2 (en) | 2018-05-18 | 2024-07-09 | Bioverativ Therapeutics Inc. | Methods of treating hemophilia A |
WO2020132297A1 (en) | 2018-12-19 | 2020-06-25 | Cue Biopharma, Inc. | Multimeric t-cell modulatory polypeptides and methods of use thereof |
WO2020181062A1 (en) | 2019-03-06 | 2020-09-10 | Cue Biopharma, Inc. | T-cell modulatory multimeric polypeptides and methods of use thereof |
WO2022032187A1 (en) | 2020-08-07 | 2022-02-10 | Bristol-Myers Squibb Company | Fgf21 combined with ccr2/5 antagonists for the treatment of fibrosis |
WO2022115597A1 (en) | 2020-11-25 | 2022-06-02 | Bristol-Myers Squibb Company | Methods of treating liver diseases |
Also Published As
Publication number | Publication date |
---|---|
US7855279B2 (en) | 2010-12-21 |
US9938331B2 (en) | 2018-04-10 |
US20120230947A1 (en) | 2012-09-13 |
US20080039341A1 (en) | 2008-02-14 |
US20120220011A1 (en) | 2012-08-30 |
US20140356326A1 (en) | 2014-12-04 |
US8492530B2 (en) | 2013-07-23 |
US20110171687A1 (en) | 2011-07-14 |
US20160280753A1 (en) | 2016-09-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9938331B2 (en) | Biologically active proteins having increased in vivo and/or in vitro stability | |
US7846445B2 (en) | Methods for production of unstructured recombinant polymers and uses thereof | |
EP1996220B1 (en) | Unstructured recombinant polymers and uses thereof | |
US20090099031A1 (en) | Genetic package and uses thereof | |
JP6175030B2 (en) | Non-structural recombinant polymers and uses thereof | |
KR102472862B1 (en) | Serum albumin-binding fibronectin type iii domains | |
US10766946B2 (en) | Fast-off rate serum albumin binding fibronectin type III domains | |
JP2010536341A (en) | Compositions and methods for altering properties of biologically active polypeptides | |
CA2644548A1 (en) | Genetic packages and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AMUNIX, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCHELLENBERGER, VOLKER;STEMMER, WILLEM P.;WANG, CHIA-WEI;AND OTHERS;SIGNING DATES FROM 20100420 TO 20100518;REEL/FRAME:024466/0508 |
|
AS | Assignment |
Owner name: AMUNIX OPERATING INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:AMUNIX, INC.;REEL/FRAME:024791/0726 Effective date: 20100708 |
|
STCB | Information on status: application discontinuation |
Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:AMUNIX PHARMACEUTICALS INC;REEL/FRAME:055220/0573 Effective date: 20210125 |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:AMUNIX PHARMACEUTICALS, INC.;REEL/FRAME:055234/0426 Effective date: 20210205 |
|
AS | Assignment |
Owner name: NIH - DEITR, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:AMUNIX PHARMACEUTICALS, INC.;REEL/FRAME:055281/0252 Effective date: 20210205 |