CN103168048B - 二聚vstm3融合蛋白和相关的组合物和方法 - Google Patents
二聚vstm3融合蛋白和相关的组合物和方法 Download PDFInfo
- Publication number
- CN103168048B CN103168048B CN201180039006.4A CN201180039006A CN103168048B CN 103168048 B CN103168048 B CN 103168048B CN 201180039006 A CN201180039006 A CN 201180039006A CN 103168048 B CN103168048 B CN 103168048B
- Authority
- CN
- China
- Prior art keywords
- seq
- cell
- polypeptide
- amino acid
- residue
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 102
- 239000000203 mixture Substances 0.000 title claims abstract description 78
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 title description 86
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 title description 80
- 108020001507 fusion proteins Proteins 0.000 title description 16
- 102000037865 fusion proteins Human genes 0.000 title description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 316
- 229920001184 polypeptide Polymers 0.000 claims abstract description 253
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 253
- 230000004927 fusion Effects 0.000 claims abstract description 124
- 210000004027 cell Anatomy 0.000 claims description 278
- 238000006471 dimerization reaction Methods 0.000 claims description 113
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 88
- 201000010099 disease Diseases 0.000 claims description 84
- 238000011282 treatment Methods 0.000 claims description 64
- 235000001014 amino acid Nutrition 0.000 claims description 61
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 55
- 102000040430 polynucleotide Human genes 0.000 claims description 30
- 108091033319 polynucleotide Proteins 0.000 claims description 30
- 239000002157 polynucleotide Substances 0.000 claims description 30
- 239000013604 expression vector Substances 0.000 claims description 28
- 125000000539 amino acid group Chemical group 0.000 claims description 27
- 230000001404 mediated effect Effects 0.000 claims description 25
- 239000003814 drug Substances 0.000 claims description 24
- 239000002253 acid Substances 0.000 claims description 22
- 201000006417 multiple sclerosis Diseases 0.000 claims description 20
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 18
- 102100029740 Poliovirus receptor Human genes 0.000 claims description 17
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 14
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 claims description 11
- 229910021529 ammonia Inorganic materials 0.000 claims description 9
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 4
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 230000005784 autoimmunity Effects 0.000 claims description 3
- 108010048507 poliovirus receptor Proteins 0.000 claims description 3
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 claims description 2
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- 210000004748 cultured cell Anatomy 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 136
- 102000004169 proteins and genes Human genes 0.000 abstract description 112
- 108010001857 Cell Surface Receptors Proteins 0.000 abstract description 37
- 239000000178 monomer Substances 0.000 abstract description 21
- 238000011160 research Methods 0.000 abstract description 15
- 102000004127 Cytokines Human genes 0.000 abstract description 9
- 108090000695 Cytokines Proteins 0.000 abstract description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- 230000000447 dimerizing effect Effects 0.000 abstract 3
- 102000006240 membrane receptors Human genes 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 131
- 239000000370 acceptor Substances 0.000 description 122
- 108020004414 DNA Proteins 0.000 description 118
- 235000018102 proteins Nutrition 0.000 description 103
- 241000699666 Mus <mouse, genus> Species 0.000 description 88
- 230000014509 gene expression Effects 0.000 description 72
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 68
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 67
- 229940024606 amino acid Drugs 0.000 description 54
- 239000002609 medium Substances 0.000 description 49
- 239000000499 gel Substances 0.000 description 46
- 150000001413 amino acids Chemical class 0.000 description 44
- 239000013612 plasmid Substances 0.000 description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 39
- 102000000844 Cell Surface Receptors Human genes 0.000 description 36
- 102000005962 receptors Human genes 0.000 description 35
- 108020003175 receptors Proteins 0.000 description 35
- 238000006243 chemical reaction Methods 0.000 description 34
- 239000002502 liposome Substances 0.000 description 32
- 238000002360 preparation method Methods 0.000 description 32
- 230000008859 change Effects 0.000 description 31
- 238000012360 testing method Methods 0.000 description 31
- 125000003729 nucleotide group Chemical group 0.000 description 30
- 230000004087 circulation Effects 0.000 description 29
- 239000002773 nucleotide Substances 0.000 description 29
- 230000000694 effects Effects 0.000 description 28
- 238000000605 extraction Methods 0.000 description 28
- 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 26
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 26
- 238000004520 electroporation Methods 0.000 description 26
- 229960000485 methotrexate Drugs 0.000 description 26
- 239000002953 phosphate buffered saline Substances 0.000 description 26
- 108060003951 Immunoglobulin Proteins 0.000 description 25
- 210000001744 T-lymphocyte Anatomy 0.000 description 25
- 102000018358 immunoglobulin Human genes 0.000 description 25
- 239000013049 sediment Substances 0.000 description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 24
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 24
- 241000588724 Escherichia coli Species 0.000 description 23
- 241001465754 Metazoa Species 0.000 description 23
- 239000002585 base Substances 0.000 description 23
- 239000000523 sample Substances 0.000 description 23
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 22
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 22
- 241000282414 Homo sapiens Species 0.000 description 21
- 235000015097 nutrients Nutrition 0.000 description 21
- 241000700605 Viruses Species 0.000 description 19
- 239000011780 sodium chloride Substances 0.000 description 19
- 239000006228 supernatant Substances 0.000 description 19
- 102000013691 Interleukin-17 Human genes 0.000 description 18
- 108050003558 Interleukin-17 Proteins 0.000 description 18
- 108010076504 Protein Sorting Signals Proteins 0.000 description 18
- 230000006870 function Effects 0.000 description 18
- 230000009182 swimming Effects 0.000 description 18
- 230000027455 binding Effects 0.000 description 17
- 102000039446 nucleic acids Human genes 0.000 description 17
- 108020004707 nucleic acids Proteins 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 235000021251 pulses Nutrition 0.000 description 17
- 230000008685 targeting Effects 0.000 description 17
- 206010033799 Paralysis Diseases 0.000 description 16
- 150000007523 nucleic acids Chemical class 0.000 description 16
- 102100038077 CD226 antigen Human genes 0.000 description 15
- 101000884298 Homo sapiens CD226 antigen Proteins 0.000 description 15
- 239000000539 dimer Substances 0.000 description 15
- 101100462378 Danio rerio otpb gene Proteins 0.000 description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- 239000005482 chemotactic factor Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 230000001629 suppression Effects 0.000 description 14
- 230000001225 therapeutic effect Effects 0.000 description 14
- 238000004043 dyeing Methods 0.000 description 13
- 239000012530 fluid Substances 0.000 description 13
- 230000004907 flux Effects 0.000 description 13
- 238000006062 fragmentation reaction Methods 0.000 description 13
- 238000011534 incubation Methods 0.000 description 13
- 238000012163 sequencing technique Methods 0.000 description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 239000008103 glucose Substances 0.000 description 12
- 238000001556 precipitation Methods 0.000 description 12
- 239000000376 reactant Substances 0.000 description 12
- 230000028327 secretion Effects 0.000 description 12
- 206010061218 Inflammation Diseases 0.000 description 11
- 241000880493 Leptailurus serval Species 0.000 description 11
- 108091034117 Oligonucleotide Proteins 0.000 description 11
- 238000012300 Sequence Analysis Methods 0.000 description 11
- 102100021164 Vasodilator-stimulated phosphoprotein Human genes 0.000 description 11
- 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 11
- -1 amino acid sequence Nucleic acid Chemical class 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 11
- 229940041514 candida albicans extract Drugs 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 11
- 210000000078 claw Anatomy 0.000 description 11
- 238000006073 displacement reaction Methods 0.000 description 11
- 108010050848 glycylleucine Proteins 0.000 description 11
- 230000004054 inflammatory process Effects 0.000 description 11
- 229910001629 magnesium chloride Inorganic materials 0.000 description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 238000001742 protein purification Methods 0.000 description 11
- 230000003248 secreting effect Effects 0.000 description 11
- 238000000926 separation method Methods 0.000 description 11
- 241000894007 species Species 0.000 description 11
- 208000024891 symptom Diseases 0.000 description 11
- 239000012137 tryptone Substances 0.000 description 11
- 108010054220 vasodilator-stimulated phosphoprotein Proteins 0.000 description 11
- 239000012138 yeast extract Substances 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 208000023275 Autoimmune disease Diseases 0.000 description 10
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 10
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 10
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 238000013467 fragmentation Methods 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 230000035772 mutation Effects 0.000 description 10
- 230000010076 replication Effects 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 238000001890 transfection Methods 0.000 description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 9
- 239000007983 Tris buffer Substances 0.000 description 9
- 206010009887 colitis Diseases 0.000 description 9
- 230000029087 digestion Effects 0.000 description 9
- 208000024908 graft versus host disease Diseases 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 210000004379 membrane Anatomy 0.000 description 9
- 239000008194 pharmaceutical composition Substances 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 9
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 9
- 241000701447 unidentified baculovirus Species 0.000 description 9
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 8
- 101150074155 DHFR gene Proteins 0.000 description 8
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 8
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 8
- 102000003814 Interleukin-10 Human genes 0.000 description 8
- 108090000174 Interleukin-10 Proteins 0.000 description 8
- 201000004681 Psoriasis Diseases 0.000 description 8
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 8
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 8
- 108010005233 alanylglutamic acid Proteins 0.000 description 8
- 230000008034 disappearance Effects 0.000 description 8
- 238000012377 drug delivery Methods 0.000 description 8
- 238000002296 dynamic light scattering Methods 0.000 description 8
- 239000013613 expression plasmid Substances 0.000 description 8
- 230000036039 immunity Effects 0.000 description 8
- 230000001976 improved effect Effects 0.000 description 8
- 239000003446 ligand Substances 0.000 description 8
- 210000003371 toe Anatomy 0.000 description 8
- 241000208340 Araliaceae Species 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 208000009386 Experimental Arthritis Diseases 0.000 description 7
- 102000004889 Interleukin-6 Human genes 0.000 description 7
- 108090001005 Interleukin-6 Proteins 0.000 description 7
- 102100026236 Interleukin-8 Human genes 0.000 description 7
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 7
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 7
- 235000003140 Panax quinquefolius Nutrition 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 210000000612 antigen-presenting cell Anatomy 0.000 description 7
- 206010003246 arthritis Diseases 0.000 description 7
- 239000006071 cream Substances 0.000 description 7
- 235000008434 ginseng Nutrition 0.000 description 7
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 7
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 230000035800 maturation Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- 235000011152 sodium sulphate Nutrition 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 210000005253 yeast cell Anatomy 0.000 description 7
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 6
- 102100023701 C-C motif chemokine 18 Human genes 0.000 description 6
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 6
- 102100025277 C-X-C motif chemokine 13 Human genes 0.000 description 6
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 description 6
- 102100034221 Growth-regulated alpha protein Human genes 0.000 description 6
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 6
- 101000858064 Homo sapiens C-X-C motif chemokine 13 Proteins 0.000 description 6
- YKZAMJXNJUWFIK-JBDRJPRFSA-N Ile-Ser-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)O)N YKZAMJXNJUWFIK-JBDRJPRFSA-N 0.000 description 6
- 102100033101 Interleukin-17B Human genes 0.000 description 6
- 102100033096 Interleukin-17D Human genes 0.000 description 6
- 108010066979 Interleukin-27 Proteins 0.000 description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 238000001042 affinity chromatography Methods 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 239000003102 growth factor Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000002744 homologous recombination Methods 0.000 description 6
- 230000006801 homologous recombination Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 125000001967 indiganyl group Chemical group [H][In]([H])[*] 0.000 description 6
- 108090000681 interleukin 20 Proteins 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 239000012139 lysis buffer Substances 0.000 description 6
- 210000004962 mammalian cell Anatomy 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000010561 standard procedure Methods 0.000 description 6
- 108010061238 threonyl-glycine Proteins 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- PPINMSZPTPRQQB-NHCYSSNCSA-N 2-[[(2s)-1-[(2s)-2-[[(2s)-2-amino-3-methylbutanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]acetic acid Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PPINMSZPTPRQQB-NHCYSSNCSA-N 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 5
- 102100023703 C-C motif chemokine 15 Human genes 0.000 description 5
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 108020004635 Complementary DNA Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 241000598860 Garcinia hanburyi Species 0.000 description 5
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 5
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 description 5
- 102000014150 Interferons Human genes 0.000 description 5
- 108010050904 Interferons Proteins 0.000 description 5
- 102100030703 Interleukin-22 Human genes 0.000 description 5
- 108090001007 Interleukin-8 Proteins 0.000 description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 5
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 5
- 241001494479 Pecora Species 0.000 description 5
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 5
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 5
- DKDHTRVDOUZZTP-IFFSRLJSSA-N Thr-Gln-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DKDHTRVDOUZZTP-IFFSRLJSSA-N 0.000 description 5
- 239000013504 Triton X-100 Substances 0.000 description 5
- 229920004890 Triton X-100 Polymers 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 108010047857 aspartylglycine Proteins 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 238000010804 cDNA synthesis Methods 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 102000003675 cytokine receptors Human genes 0.000 description 5
- 108010057085 cytokine receptors Proteins 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 229940117709 gamboge Drugs 0.000 description 5
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 5
- 238000001415 gene therapy Methods 0.000 description 5
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 5
- 239000008187 granular material Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 230000003993 interaction Effects 0.000 description 5
- 229940079322 interferon Drugs 0.000 description 5
- 108010074109 interleukin-22 Proteins 0.000 description 5
- 229940096397 interleukin-8 Drugs 0.000 description 5
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 108010026333 seryl-proline Proteins 0.000 description 5
- 238000013456 study Methods 0.000 description 5
- 108010072644 valyl-alanyl-prolyl-glycine Proteins 0.000 description 5
- 230000003442 weekly effect Effects 0.000 description 5
- FOHXUHGZZKETFI-JBDRJPRFSA-N Ala-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C)N FOHXUHGZZKETFI-JBDRJPRFSA-N 0.000 description 4
- 101710153593 Albumin A Proteins 0.000 description 4
- OFIYLHVAAJYRBC-HJWJTTGWSA-N Arg-Ile-Phe Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](Cc1ccccc1)C(O)=O OFIYLHVAAJYRBC-HJWJTTGWSA-N 0.000 description 4
- RZVVKNIACROXRM-ZLUOBGJFSA-N Asn-Ala-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N RZVVKNIACROXRM-ZLUOBGJFSA-N 0.000 description 4
- AYFVRYXNDHBECD-YUMQZZPRSA-N Asp-Leu-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O AYFVRYXNDHBECD-YUMQZZPRSA-N 0.000 description 4
- 102100036841 C-C motif chemokine 1 Human genes 0.000 description 4
- 102100023702 C-C motif chemokine 13 Human genes 0.000 description 4
- 102100023705 C-C motif chemokine 14 Human genes 0.000 description 4
- 102100023700 C-C motif chemokine 16 Human genes 0.000 description 4
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 4
- 102100036850 C-C motif chemokine 23 Human genes 0.000 description 4
- 102100036849 C-C motif chemokine 24 Human genes 0.000 description 4
- 102100021933 C-C motif chemokine 25 Human genes 0.000 description 4
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 4
- 102100032366 C-C motif chemokine 7 Human genes 0.000 description 4
- 102100034871 C-C motif chemokine 8 Human genes 0.000 description 4
- 102100031658 C-X-C chemokine receptor type 5 Human genes 0.000 description 4
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 4
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 description 4
- 102100036153 C-X-C motif chemokine 6 Human genes 0.000 description 4
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 description 4
- 108091006020 Fc-tagged proteins Proteins 0.000 description 4
- ZRXBYKAOFHLTDN-GUBZILKMSA-N Gln-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(=O)N)N ZRXBYKAOFHLTDN-GUBZILKMSA-N 0.000 description 4
- QYKBTDOAMKORGL-FXQIFTODSA-N Gln-Gln-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N QYKBTDOAMKORGL-FXQIFTODSA-N 0.000 description 4
- PMSDOVISAARGAV-FHWLQOOXSA-N Glu-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCC(O)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 PMSDOVISAARGAV-FHWLQOOXSA-N 0.000 description 4
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 4
- 102100020948 Growth hormone receptor Human genes 0.000 description 4
- ORERHHPZDDEMSC-VGDYDELISA-N His-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ORERHHPZDDEMSC-VGDYDELISA-N 0.000 description 4
- 101000978381 Homo sapiens C-C motif chemokine 14 Proteins 0.000 description 4
- 101000978375 Homo sapiens C-C motif chemokine 16 Proteins 0.000 description 4
- 101000978371 Homo sapiens C-C motif chemokine 18 Proteins 0.000 description 4
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 4
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 4
- 101000713081 Homo sapiens C-C motif chemokine 23 Proteins 0.000 description 4
- 101000713078 Homo sapiens C-C motif chemokine 24 Proteins 0.000 description 4
- 101000897486 Homo sapiens C-C motif chemokine 25 Proteins 0.000 description 4
- 101000922405 Homo sapiens C-X-C chemokine receptor type 5 Proteins 0.000 description 4
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 description 4
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 description 4
- 101000853000 Homo sapiens Interleukin-26 Proteins 0.000 description 4
- 101000947178 Homo sapiens Platelet basic protein Proteins 0.000 description 4
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 4
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 4
- YGDWPQCLFJNMOL-MNXVOIDGSA-N Ile-Leu-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YGDWPQCLFJNMOL-MNXVOIDGSA-N 0.000 description 4
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 4
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 4
- 102000003815 Interleukin-11 Human genes 0.000 description 4
- 108090000177 Interleukin-11 Proteins 0.000 description 4
- 108010065805 Interleukin-12 Proteins 0.000 description 4
- 102000003812 Interleukin-15 Human genes 0.000 description 4
- 108090000172 Interleukin-15 Proteins 0.000 description 4
- 101800003050 Interleukin-16 Proteins 0.000 description 4
- 102100033105 Interleukin-17C Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102100036679 Interleukin-26 Human genes 0.000 description 4
- 108010002386 Interleukin-3 Proteins 0.000 description 4
- 108090000978 Interleukin-4 Proteins 0.000 description 4
- 108010002586 Interleukin-7 Proteins 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- JRJLGNFWYFSJHB-HOCLYGCPSA-N Leu-Gly-Trp Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O JRJLGNFWYFSJHB-HOCLYGCPSA-N 0.000 description 4
- 101000946797 Mus musculus C-C motif chemokine 9 Proteins 0.000 description 4
- 108010025020 Nerve Growth Factor Proteins 0.000 description 4
- OQTDZEJJWWAGJT-KKUMJFAQSA-N Phe-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O OQTDZEJJWWAGJT-KKUMJFAQSA-N 0.000 description 4
- 102000002808 Pituitary adenylate cyclase-activating polypeptide Human genes 0.000 description 4
- 108010004684 Pituitary adenylate cyclase-activating polypeptide Proteins 0.000 description 4
- 102100036154 Platelet basic protein Human genes 0.000 description 4
- 102100030304 Platelet factor 4 Human genes 0.000 description 4
- 208000007400 Relapsing-Remitting Multiple Sclerosis Diseases 0.000 description 4
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 4
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 4
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 4
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 4
- 108010068542 Somatotropin Receptors Proteins 0.000 description 4
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 4
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 4
- AQAMPXBRJJWPNI-JHEQGTHGSA-N Thr-Gly-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AQAMPXBRJJWPNI-JHEQGTHGSA-N 0.000 description 4
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 4
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 4
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 4
- UPODKYBYUBTWSV-BZSNNMDCSA-N Tyr-Phe-Cys Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CS)C(O)=O)C1=CC=C(O)C=C1 UPODKYBYUBTWSV-BZSNNMDCSA-N 0.000 description 4
- XJPXTYLVMUZGNW-IHRRRGAJSA-N Tyr-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O XJPXTYLVMUZGNW-IHRRRGAJSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 150000001412 amines Chemical class 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 244000309464 bull Species 0.000 description 4
- 208000007118 chronic progressive multiple sclerosis Diseases 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 108010074108 interleukin-21 Proteins 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000000116 mitigating effect Effects 0.000 description 4
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 206010063401 primary progressive multiple sclerosis Diseases 0.000 description 4
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 4
- 230000001185 psoriatic effect Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- ADNPLDHMAVUMIW-CUZNLEPHSA-N substance P Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)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](N)CCCN=C(N)N)C1=CC=CC=C1 ADNPLDHMAVUMIW-CUZNLEPHSA-N 0.000 description 4
- 238000007492 two-way ANOVA Methods 0.000 description 4
- 108010037335 tyrosyl-prolyl-glycyl-glycine Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 3
- YIGLXQRFQVWFEY-NRPADANISA-N Ala-Gln-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O YIGLXQRFQVWFEY-NRPADANISA-N 0.000 description 3
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 3
- BVSGPHDECMJBDE-HGNGGELXSA-N Ala-Glu-His Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N BVSGPHDECMJBDE-HGNGGELXSA-N 0.000 description 3
- ZBLQIYPCUWZSRZ-QEJZJMRPSA-N Ala-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CC1=CC=CC=C1 ZBLQIYPCUWZSRZ-QEJZJMRPSA-N 0.000 description 3
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 3
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 3
- 239000012114 Alexa Fluor 647 Substances 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 3
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 3
- SEKBHZJLARBNPB-GHCJXIJMSA-N Asn-Ile-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O SEKBHZJLARBNPB-GHCJXIJMSA-N 0.000 description 3
- FHCRKXCTKSHNOE-QEJZJMRPSA-N Asn-Trp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N FHCRKXCTKSHNOE-QEJZJMRPSA-N 0.000 description 3
- XYBJLTKSGFBLCS-QXEWZRGKSA-N Asp-Arg-Val Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC(O)=O XYBJLTKSGFBLCS-QXEWZRGKSA-N 0.000 description 3
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 3
- NAAAPCLFJPURAM-HJGDQZAQSA-N Asp-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N)O NAAAPCLFJPURAM-HJGDQZAQSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 3
- 102100021942 C-C motif chemokine 28 Human genes 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 208000011231 Crohn disease Diseases 0.000 description 3
- RRJOQIBQVZDVCW-SRVKXCTJSA-N Cys-His-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CS)N RRJOQIBQVZDVCW-SRVKXCTJSA-N 0.000 description 3
- DRXOWZZHCSBUOI-YJRXYDGGSA-N Cys-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CS)N)O DRXOWZZHCSBUOI-YJRXYDGGSA-N 0.000 description 3
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 3
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 3
- OCJRHJZKGGSPRW-IUCAKERBSA-N Glu-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O OCJRHJZKGGSPRW-IUCAKERBSA-N 0.000 description 3
- VNCNWQPIQYAMAK-ACZMJKKPSA-N Glu-Ser-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O VNCNWQPIQYAMAK-ACZMJKKPSA-N 0.000 description 3
- 229920001503 Glucan Polymers 0.000 description 3
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-N 0.000 description 3
- OCQUNKSFDYDXBG-QXEWZRGKSA-N Gly-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OCQUNKSFDYDXBG-QXEWZRGKSA-N 0.000 description 3
- XMPXVJIDADUOQB-RCOVLWMOSA-N Gly-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C([O-])=O)NC(=O)CNC(=O)C[NH3+] XMPXVJIDADUOQB-RCOVLWMOSA-N 0.000 description 3
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 3
- VNNRLUNBJSWZPF-ZKWXMUAHSA-N Gly-Ser-Ile Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VNNRLUNBJSWZPF-ZKWXMUAHSA-N 0.000 description 3
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 208000009329 Graft vs Host Disease Diseases 0.000 description 3
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 3
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 3
- 208000015023 Graves' disease Diseases 0.000 description 3
- 108010051696 Growth Hormone Proteins 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 101000897477 Homo sapiens C-C motif chemokine 28 Proteins 0.000 description 3
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 3
- JSLIXOUMAOUGBN-JUKXBJQTSA-N Ile-Tyr-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N JSLIXOUMAOUGBN-JUKXBJQTSA-N 0.000 description 3
- 108090000176 Interleukin-13 Proteins 0.000 description 3
- 108010002616 Interleukin-5 Proteins 0.000 description 3
- 108010002335 Interleukin-9 Proteins 0.000 description 3
- 241000235058 Komagataella pastoris Species 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 description 3
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 3
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 3
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 3
- NTXYXFDMIHXTHE-WDSOQIARSA-N Leu-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 NTXYXFDMIHXTHE-WDSOQIARSA-N 0.000 description 3
- MRWXLRGAFDOILG-DCAQKATOSA-N Lys-Gln-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MRWXLRGAFDOILG-DCAQKATOSA-N 0.000 description 3
- WXJXYMFUTRXRGO-UWVGGRQHSA-N Met-His-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CNC=N1 WXJXYMFUTRXRGO-UWVGGRQHSA-N 0.000 description 3
- CIIJWIAORKTXAH-FJXKBIBVSA-N Met-Thr-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O CIIJWIAORKTXAH-FJXKBIBVSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 241001452677 Ogataea methanolica Species 0.000 description 3
- 102100040557 Osteopontin Human genes 0.000 description 3
- 108010081689 Osteopontin Proteins 0.000 description 3
- 108010081690 Pertussis Toxin Proteins 0.000 description 3
- RJYBHZVWJPUSLB-QEWYBTABSA-N Phe-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N RJYBHZVWJPUSLB-QEWYBTABSA-N 0.000 description 3
- NKLDZIPTGKBDBB-HTUGSXCWSA-N Phe-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N)O NKLDZIPTGKBDBB-HTUGSXCWSA-N 0.000 description 3
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 description 3
- LNICFEXCAHIJOR-DCAQKATOSA-N Pro-Ser-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LNICFEXCAHIJOR-DCAQKATOSA-N 0.000 description 3
- UAJAYRMZGNQILN-BQBZGAKWSA-N Ser-Gly-Met Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O UAJAYRMZGNQILN-BQBZGAKWSA-N 0.000 description 3
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 3
- 102100038803 Somatotropin Human genes 0.000 description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- FQPQPTHMHZKGFM-XQXXSGGOSA-N Thr-Ala-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O FQPQPTHMHZKGFM-XQXXSGGOSA-N 0.000 description 3
- XTCNBOBTROGWMW-RWRJDSDZSA-N Thr-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XTCNBOBTROGWMW-RWRJDSDZSA-N 0.000 description 3
- PCMDGXKXVMBIFP-VEVYYDQMSA-N Thr-Met-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMDGXKXVMBIFP-VEVYYDQMSA-N 0.000 description 3
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 3
- DIHPMRTXPYMDJZ-KAOXEZKKSA-N Thr-Tyr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N)O DIHPMRTXPYMDJZ-KAOXEZKKSA-N 0.000 description 3
- 206010052779 Transplant rejections Diseases 0.000 description 3
- QEJHHFFFCUDPDV-WDSOQIARSA-N Trp-His-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N QEJHHFFFCUDPDV-WDSOQIARSA-N 0.000 description 3
- CCZXBOFIBYQLEV-IHPCNDPISA-N Trp-Leu-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(O)=O CCZXBOFIBYQLEV-IHPCNDPISA-N 0.000 description 3
- 102100040247 Tumor necrosis factor Human genes 0.000 description 3
- VTCKHZJKWQENKX-KBPBESRZSA-N Tyr-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O VTCKHZJKWQENKX-KBPBESRZSA-N 0.000 description 3
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 3
- 241000700618 Vaccinia virus Species 0.000 description 3
- IZFVRRYRMQFVGX-NRPADANISA-N Val-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N IZFVRRYRMQFVGX-NRPADANISA-N 0.000 description 3
- AUMNPAUHKUNHHN-BYULHYEWSA-N Val-Asn-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N AUMNPAUHKUNHHN-BYULHYEWSA-N 0.000 description 3
- XKVXSCHXGJOQND-ZOBUZTSGSA-N Val-Asp-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N XKVXSCHXGJOQND-ZOBUZTSGSA-N 0.000 description 3
- XEYUMGGWQCIWAR-XVKPBYJWSA-N Val-Gln-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N XEYUMGGWQCIWAR-XVKPBYJWSA-N 0.000 description 3
- FTKXYXACXYOHND-XUXIUFHCSA-N Val-Ile-Leu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O FTKXYXACXYOHND-XUXIUFHCSA-N 0.000 description 3
- KISFXYYRKKNLOP-IHRRRGAJSA-N Val-Phe-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N KISFXYYRKKNLOP-IHRRRGAJSA-N 0.000 description 3
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 3
- UQMPYVLTQCGRSK-IFFSRLJSSA-N Val-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N)O UQMPYVLTQCGRSK-IFFSRLJSSA-N 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000000172 allergic effect Effects 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 210000003423 ankle Anatomy 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108010092854 aspartyllysine Proteins 0.000 description 3
- 208000010668 atopic eczema Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000010256 biochemical assay Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000012501 chromatography medium Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 201000005332 contagious pustular dermatitis Diseases 0.000 description 3
- 238000013270 controlled release Methods 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009792 diffusion process Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000006334 disulfide bridging Effects 0.000 description 3
- 206010014599 encephalitis Diseases 0.000 description 3
- 201000002491 encephalomyelitis Diseases 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 239000003797 essential amino acid Substances 0.000 description 3
- 235000020776 essential amino acid Nutrition 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 108010067216 glycyl-glycyl-glycine Proteins 0.000 description 3
- 108010084389 glycyltryptophan Proteins 0.000 description 3
- 239000000122 growth hormone Substances 0.000 description 3
- 108010036413 histidylglycine Proteins 0.000 description 3
- 108010092114 histidylphenylalanine Proteins 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 208000026278 immune system disease Diseases 0.000 description 3
- 239000007943 implant Substances 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108010064235 lysylglycine Proteins 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 108010085203 methionylmethionine Proteins 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 231100000219 mutagenic Toxicity 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108010070643 prolylglutamic acid Proteins 0.000 description 3
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 108010029384 tryptophyl-histidine Proteins 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 210000000707 wrist Anatomy 0.000 description 3
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-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
- 108020005029 5' Flanking Region Proteins 0.000 description 2
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 2
- 208000002874 Acne Vulgaris Diseases 0.000 description 2
- 102100022455 Adrenocorticotropic hormone receptor Human genes 0.000 description 2
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 2
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 2
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 102000005590 Anaphylatoxin C5a Receptor Human genes 0.000 description 2
- 108010059426 Anaphylatoxin C5a Receptor Proteins 0.000 description 2
- 102400000068 Angiostatin Human genes 0.000 description 2
- 108010079709 Angiostatins Proteins 0.000 description 2
- 101100339431 Arabidopsis thaliana HMGB2 gene Proteins 0.000 description 2
- VEAIMHJZTIDCIH-KKUMJFAQSA-N Arg-Phe-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VEAIMHJZTIDCIH-KKUMJFAQSA-N 0.000 description 2
- XQQVCUIBGYFKDC-OLHMAJIHSA-N Asn-Asp-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XQQVCUIBGYFKDC-OLHMAJIHSA-N 0.000 description 2
- HNXWVVHIGTZTBO-LKXGYXEUSA-N Asn-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O HNXWVVHIGTZTBO-LKXGYXEUSA-N 0.000 description 2
- SNDBKTFJWVEVPO-WHFBIAKZSA-N Asp-Gly-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SNDBKTFJWVEVPO-WHFBIAKZSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 2
- 102100022718 Atypical chemokine receptor 2 Human genes 0.000 description 2
- 206010069002 Autoimmune pancreatitis Diseases 0.000 description 2
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 2
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 2
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 2
- 102100025218 B-cell differentiation antigen CD72 Human genes 0.000 description 2
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 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 2
- 241000894006 Bacteria Species 0.000 description 2
- 102000001893 Bone Morphogenetic Protein Receptors Human genes 0.000 description 2
- 108010040422 Bone Morphogenetic Protein Receptors Proteins 0.000 description 2
- 102100031172 C-C chemokine receptor type 1 Human genes 0.000 description 2
- 101710149814 C-C chemokine receptor type 1 Proteins 0.000 description 2
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 description 2
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 description 2
- 102100024167 C-C chemokine receptor type 3 Human genes 0.000 description 2
- 101710149862 C-C chemokine receptor type 3 Proteins 0.000 description 2
- 102100037853 C-C chemokine receptor type 4 Human genes 0.000 description 2
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 2
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 2
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 2
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 2
- 102100036305 C-C chemokine receptor type 8 Human genes 0.000 description 2
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 description 2
- 101710155835 C-C motif chemokine 1 Proteins 0.000 description 2
- 101710112613 C-C motif chemokine 13 Proteins 0.000 description 2
- 102100023698 C-C motif chemokine 17 Human genes 0.000 description 2
- 101710112622 C-C motif chemokine 19 Proteins 0.000 description 2
- 102100021936 C-C motif chemokine 27 Human genes 0.000 description 2
- 101710155834 C-C motif chemokine 7 Proteins 0.000 description 2
- 101710155833 C-C motif chemokine 8 Proteins 0.000 description 2
- 102100036166 C-X-C chemokine receptor type 1 Human genes 0.000 description 2
- 102100028989 C-X-C chemokine receptor type 2 Human genes 0.000 description 2
- 102100028990 C-X-C chemokine receptor type 3 Human genes 0.000 description 2
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 2
- 101710098275 C-X-C motif chemokine 10 Proteins 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
- 102100036189 C-X-C motif chemokine 3 Human genes 0.000 description 2
- 101710085504 C-X-C motif chemokine 6 Proteins 0.000 description 2
- 101710085500 C-X-C motif chemokine 9 Proteins 0.000 description 2
- 101150073986 C3AR1 gene Proteins 0.000 description 2
- 101150049756 CCL6 gene Proteins 0.000 description 2
- 101150011672 CCL9 gene Proteins 0.000 description 2
- 102100027207 CD27 antigen Human genes 0.000 description 2
- 108010029697 CD40 Ligand Proteins 0.000 description 2
- 101150013553 CD40 gene Proteins 0.000 description 2
- 102100032937 CD40 ligand Human genes 0.000 description 2
- 102100025221 CD70 antigen Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 2
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 2
- 102100038518 Calcitonin Human genes 0.000 description 2
- 108060001064 Calcitonin Proteins 0.000 description 2
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 description 2
- 241000222128 Candida maltosa Species 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102100038608 Cathelicidin antimicrobial peptide Human genes 0.000 description 2
- 101710140438 Cathelicidin antimicrobial peptide Proteins 0.000 description 2
- 101150075117 Ccl12 gene Proteins 0.000 description 2
- 108010082155 Chemokine CCL18 Proteins 0.000 description 2
- 108010082161 Chemokine CCL19 Proteins 0.000 description 2
- 102000003805 Chemokine CCL19 Human genes 0.000 description 2
- 108010083702 Chemokine CCL21 Proteins 0.000 description 2
- 108010055165 Chemokine CCL4 Proteins 0.000 description 2
- 102000001326 Chemokine CCL4 Human genes 0.000 description 2
- 108010055166 Chemokine CCL5 Proteins 0.000 description 2
- 108010078239 Chemokine CX3CL1 Proteins 0.000 description 2
- 102100035294 Chemokine XC receptor 1 Human genes 0.000 description 2
- 101710172503 Chemokine-binding protein Proteins 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 208000023355 Chronic beryllium disease Diseases 0.000 description 2
- 206010009900 Colitis ulcerative Diseases 0.000 description 2
- 102400000498 Connective tissue-activating peptide III Human genes 0.000 description 2
- 102400000739 Corticotropin Human genes 0.000 description 2
- 101800000414 Corticotropin Proteins 0.000 description 2
- 108010074311 Corticotropin Receptors Proteins 0.000 description 2
- 239000000055 Corticotropin-Releasing Hormone Substances 0.000 description 2
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010002069 Defensins Proteins 0.000 description 2
- 102000000541 Defensins Human genes 0.000 description 2
- 102400001047 Endostatin Human genes 0.000 description 2
- 108010079505 Endostatins Proteins 0.000 description 2
- 102100023688 Eotaxin Human genes 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 102000013818 Fractalkine Human genes 0.000 description 2
- 102100020997 Fractalkine Human genes 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 2
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 2
- 102000030782 GTP binding Human genes 0.000 description 2
- 108091000058 GTP-Binding Proteins 0.000 description 2
- 101710115997 Gamma-tubulin complex component 2 Proteins 0.000 description 2
- RKAQZCDMSUQTSS-FXQIFTODSA-N Gln-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N RKAQZCDMSUQTSS-FXQIFTODSA-N 0.000 description 2
- DHNWZLGBTPUTQQ-QEJZJMRPSA-N Gln-Asp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N DHNWZLGBTPUTQQ-QEJZJMRPSA-N 0.000 description 2
- YRHZWVKUFWCEPW-GLLZPBPUSA-N Gln-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O YRHZWVKUFWCEPW-GLLZPBPUSA-N 0.000 description 2
- ICRKQMRFXYDYMK-LAEOZQHASA-N Gln-Val-Asn Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ICRKQMRFXYDYMK-LAEOZQHASA-N 0.000 description 2
- HNAUFGBKJLTWQE-IFFSRLJSSA-N Gln-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(=O)N)N)O HNAUFGBKJLTWQE-IFFSRLJSSA-N 0.000 description 2
- 206010018364 Glomerulonephritis Diseases 0.000 description 2
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 2
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 description 2
- XIKYNVKEUINBGL-IUCAKERBSA-N Glu-His-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O XIKYNVKEUINBGL-IUCAKERBSA-N 0.000 description 2
- ZALGPUWUVHOGAE-GVXVVHGQSA-N Glu-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)O)N ZALGPUWUVHOGAE-GVXVVHGQSA-N 0.000 description 2
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 2
- HFXJIZNEXNIZIJ-BQBZGAKWSA-N Gly-Glu-Gln Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFXJIZNEXNIZIJ-BQBZGAKWSA-N 0.000 description 2
- CUYLIWAAAYJKJH-RYUDHWBXSA-N Gly-Glu-Tyr Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CUYLIWAAAYJKJH-RYUDHWBXSA-N 0.000 description 2
- RUDRIZRGOLQSMX-IUCAKERBSA-N Gly-Met-Met Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCSC)C(O)=O RUDRIZRGOLQSMX-IUCAKERBSA-N 0.000 description 2
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 2
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 2
- FOKISINOENBSDM-WLTAIBSBSA-N Gly-Thr-Tyr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FOKISINOENBSDM-WLTAIBSBSA-N 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 208000003807 Graves Disease Diseases 0.000 description 2
- 102000009465 Growth Factor Receptors Human genes 0.000 description 2
- 108010009202 Growth Factor Receptors Proteins 0.000 description 2
- 108700010013 HMGB1 Proteins 0.000 description 2
- 101150021904 HMGB1 gene Proteins 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 208000001204 Hashimoto Disease Diseases 0.000 description 2
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 2
- 208000035186 Hemolytic Autoimmune Anemia Diseases 0.000 description 2
- 102100032813 Hepatocyte growth factor-like protein Human genes 0.000 description 2
- 241000175212 Herpesvirales Species 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101000728693 Homo sapiens 28S ribosomal protein S11, mitochondrial Proteins 0.000 description 2
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 2
- 101000678892 Homo sapiens Atypical chemokine receptor 2 Proteins 0.000 description 2
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 2
- 101000934359 Homo sapiens B-cell differentiation antigen CD72 Proteins 0.000 description 2
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 description 2
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 2
- 101000716063 Homo sapiens C-C chemokine receptor type 8 Proteins 0.000 description 2
- 101000716070 Homo sapiens C-C chemokine receptor type 9 Proteins 0.000 description 2
- 101000978379 Homo sapiens C-C motif chemokine 13 Proteins 0.000 description 2
- 101000978362 Homo sapiens C-C motif chemokine 17 Proteins 0.000 description 2
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 description 2
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 2
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 description 2
- 101000946794 Homo sapiens C-C motif chemokine 8 Proteins 0.000 description 2
- 101000947174 Homo sapiens C-X-C chemokine receptor type 1 Proteins 0.000 description 2
- 101000916050 Homo sapiens C-X-C chemokine receptor type 3 Proteins 0.000 description 2
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 2
- 101000858088 Homo sapiens C-X-C motif chemokine 10 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
- 101000889128 Homo sapiens C-X-C motif chemokine 2 Proteins 0.000 description 2
- 101000947193 Homo sapiens C-X-C motif chemokine 3 Proteins 0.000 description 2
- 101000947177 Homo sapiens C-X-C motif chemokine 6 Proteins 0.000 description 2
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 description 2
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 2
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 2
- 101000804783 Homo sapiens Chemokine XC receptor 1 Proteins 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- 101000978392 Homo sapiens Eotaxin Proteins 0.000 description 2
- 101000854520 Homo sapiens Fractalkine Proteins 0.000 description 2
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 2
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 2
- 101001017968 Homo sapiens Leukotriene B4 receptor 1 Proteins 0.000 description 2
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 description 2
- 101100460850 Homo sapiens NCR3LG1 gene Proteins 0.000 description 2
- 101000973997 Homo sapiens Nucleosome assembly protein 1-like 4 Proteins 0.000 description 2
- 101000582950 Homo sapiens Platelet factor 4 Proteins 0.000 description 2
- 101000831616 Homo sapiens Protachykinin-1 Proteins 0.000 description 2
- 101000668165 Homo sapiens RNA-binding motif, single-stranded-interacting protein 1 Proteins 0.000 description 2
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 2
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 2
- 101000610602 Homo sapiens Tumor necrosis factor receptor superfamily member 10C Proteins 0.000 description 2
- 101000610609 Homo sapiens Tumor necrosis factor receptor superfamily member 10D Proteins 0.000 description 2
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 2
- 101000801227 Homo sapiens Tumor necrosis factor receptor superfamily member 19 Proteins 0.000 description 2
- 101000597785 Homo sapiens Tumor necrosis factor receptor superfamily member 6B Proteins 0.000 description 2
- 101000818522 Homo sapiens fMet-Leu-Phe receptor Proteins 0.000 description 2
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 2
- XLXPYSDGMXTTNQ-DKIMLUQUSA-N Ile-Phe-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(O)=O XLXPYSDGMXTTNQ-DKIMLUQUSA-N 0.000 description 2
- 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 2
- 206010061598 Immunodeficiency Diseases 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 108010008212 Integrin alpha4beta1 Proteins 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 102100037971 Interferon lambda receptor 1 Human genes 0.000 description 2
- 101710145151 Interferon lambda receptor 1 Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- 108090000467 Interferon-beta Proteins 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 108010065637 Interleukin-23 Proteins 0.000 description 2
- 102100036705 Interleukin-23 subunit alpha Human genes 0.000 description 2
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 2
- 108010018951 Interleukin-8B Receptors Proteins 0.000 description 2
- 208000007766 Kaposi sarcoma Diseases 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical group SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 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
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 102000016267 Leptin Human genes 0.000 description 2
- 108010092277 Leptin Proteins 0.000 description 2
- PVMPDMIKUVNOBD-CIUDSAMLSA-N Leu-Asp-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O PVMPDMIKUVNOBD-CIUDSAMLSA-N 0.000 description 2
- DXYBNWJZJVSZAE-GUBZILKMSA-N Leu-Gln-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N DXYBNWJZJVSZAE-GUBZILKMSA-N 0.000 description 2
- GPICTNQYKHHHTH-GUBZILKMSA-N Leu-Gln-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GPICTNQYKHHHTH-GUBZILKMSA-N 0.000 description 2
- WQWSMEOYXJTFRU-GUBZILKMSA-N Leu-Glu-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O WQWSMEOYXJTFRU-GUBZILKMSA-N 0.000 description 2
- HVJVUYQWFYMGJS-GVXVVHGQSA-N Leu-Glu-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVJVUYQWFYMGJS-GVXVVHGQSA-N 0.000 description 2
- AUNMOHYWTAPQLA-XUXIUFHCSA-N Leu-Met-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AUNMOHYWTAPQLA-XUXIUFHCSA-N 0.000 description 2
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 2
- 102100033374 Leukotriene B4 receptor 1 Human genes 0.000 description 2
- 229930184725 Lipoxin Natural products 0.000 description 2
- 102100035304 Lymphotactin Human genes 0.000 description 2
- 108010091221 Lymphotoxin beta Receptor Proteins 0.000 description 2
- 102000018170 Lymphotoxin beta Receptor Human genes 0.000 description 2
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 2
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 2
- 102100026894 Lymphotoxin-beta Human genes 0.000 description 2
- 108090000362 Lymphotoxin-beta Proteins 0.000 description 2
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 2
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 2
- 102100027754 Mast/stem cell growth factor receptor Kit Human genes 0.000 description 2
- 102400000740 Melanocyte-stimulating hormone alpha Human genes 0.000 description 2
- 101710200814 Melanotropin alpha Proteins 0.000 description 2
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 2
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 101000978374 Mus musculus C-C motif chemokine 12 Proteins 0.000 description 2
- 101100005550 Mus musculus Ccl1 gene Proteins 0.000 description 2
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- PRQROPMIIGLWRP-UHFFFAOYSA-N N-formyl-methionyl-leucyl-phenylalanin Chemical compound CSCCC(NC=O)C(=O)NC(CC(C)C)C(=O)NC(C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-UHFFFAOYSA-N 0.000 description 2
- 108091061960 Naked DNA Proteins 0.000 description 2
- 108010004222 Natural Cytotoxicity Triggering Receptor 3 Proteins 0.000 description 2
- 102100032852 Natural cytotoxicity triggering receptor 3 Human genes 0.000 description 2
- 102100029527 Natural cytotoxicity triggering receptor 3 ligand 1 Human genes 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 description 2
- 206010029240 Neuritis Diseases 0.000 description 2
- 241000221960 Neurospora Species 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 102000004140 Oncostatin M Human genes 0.000 description 2
- 108090000630 Oncostatin M Proteins 0.000 description 2
- 108010082522 Oncostatin M Receptors Proteins 0.000 description 2
- 102100030098 Oncostatin-M-specific receptor subunit beta Human genes 0.000 description 2
- 108010035042 Osteoprotegerin Proteins 0.000 description 2
- 102000008108 Osteoprotegerin Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000700639 Parapoxvirus Species 0.000 description 2
- 208000031845 Pernicious anaemia Diseases 0.000 description 2
- JDMKQHSHKJHAHR-UHFFFAOYSA-N Phe-Phe-Leu-Tyr Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)CC1=CC=CC=C1 JDMKQHSHKJHAHR-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 108010003541 Platelet Activating Factor Proteins 0.000 description 2
- 108090000778 Platelet factor 4 Proteins 0.000 description 2
- 108700023400 Platelet-activating factor receptors Proteins 0.000 description 2
- 102100040681 Platelet-derived growth factor C Human genes 0.000 description 2
- 102100040682 Platelet-derived growth factor D Human genes 0.000 description 2
- 101710170209 Platelet-derived growth factor D Proteins 0.000 description 2
- 102100037596 Platelet-derived growth factor subunit A Human genes 0.000 description 2
- CGBYDGAJHSOGFQ-LPEHRKFASA-N Pro-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 CGBYDGAJHSOGFQ-LPEHRKFASA-N 0.000 description 2
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 2
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 2
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 description 2
- KBUAPZAZPWNYSW-SRVKXCTJSA-N Pro-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KBUAPZAZPWNYSW-SRVKXCTJSA-N 0.000 description 2
- 102100024819 Prolactin Human genes 0.000 description 2
- 108010057464 Prolactin Proteins 0.000 description 2
- 102100024304 Protachykinin-1 Human genes 0.000 description 2
- 102100029812 Protein S100-A12 Human genes 0.000 description 2
- 206010049680 Quadriparesis Diseases 0.000 description 2
- 206010037714 Quadriplegia Diseases 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 238000011579 SCID mouse model Methods 0.000 description 2
- 101100209842 Saimiriine herpesvirus 2 (strain 11) 74 gene Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- BTPAWKABYQMKKN-LKXGYXEUSA-N Ser-Asp-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BTPAWKABYQMKKN-LKXGYXEUSA-N 0.000 description 2
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 2
- DJACUBDEDBZKLQ-KBIXCLLPSA-N Ser-Ile-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O DJACUBDEDBZKLQ-KBIXCLLPSA-N 0.000 description 2
- HBTCFCHYALPXME-HTFCKZLJSA-N Ser-Ile-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HBTCFCHYALPXME-HTFCKZLJSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 229940122055 Serine protease inhibitor Drugs 0.000 description 2
- 101710102218 Serine protease inhibitor Proteins 0.000 description 2
- 101710181917 Serine proteinase inhibitor 1 Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 208000021386 Sjogren Syndrome Diseases 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 241000256251 Spodoptera frugiperda Species 0.000 description 2
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 2
- 101150056647 TNFRSF4 gene Proteins 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 2
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 2
- LVRFMARKDGGZMX-IZPVPAKOSA-N Thr-Tyr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=C(O)C=C1 LVRFMARKDGGZMX-IZPVPAKOSA-N 0.000 description 2
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 2
- 241000255993 Trichoplusia ni Species 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 102100040115 Tumor necrosis factor receptor superfamily member 10C Human genes 0.000 description 2
- 102100040110 Tumor necrosis factor receptor superfamily member 10D Human genes 0.000 description 2
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 2
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 2
- 101710178300 Tumor necrosis factor receptor superfamily member 13C Proteins 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
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 2
- 102100035284 Tumor necrosis factor receptor superfamily member 6B Human genes 0.000 description 2
- 102100032807 Tumor necrosis factor-inducible gene 6 protein Human genes 0.000 description 2
- 101710169430 Tumor necrosis factor-inducible gene 6 protein Proteins 0.000 description 2
- IIJWXEUNETVJPV-IHRRRGAJSA-N Tyr-Arg-Ser Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N)O IIJWXEUNETVJPV-IHRRRGAJSA-N 0.000 description 2
- NHOVZGFNTGMYMI-KKUMJFAQSA-N Tyr-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 NHOVZGFNTGMYMI-KKUMJFAQSA-N 0.000 description 2
- 101150044134 US28 gene Proteins 0.000 description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 description 2
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 2
- SBJCTAZFSZXWSR-AVGNSLFASA-N Val-Met-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N SBJCTAZFSZXWSR-AVGNSLFASA-N 0.000 description 2
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 2
- ZLNYBMWGPOKSLW-LSJOCFKGSA-N Val-Val-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLNYBMWGPOKSLW-LSJOCFKGSA-N 0.000 description 2
- LLJLBRRXKZTTRD-GUBZILKMSA-N Val-Val-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)O)N LLJLBRRXKZTTRD-GUBZILKMSA-N 0.000 description 2
- 206010047642 Vitiligo Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 206010000496 acne Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000001467 acupuncture Methods 0.000 description 2
- 108010044940 alanylglutamine Proteins 0.000 description 2
- 125000003282 alkyl amino group Chemical group 0.000 description 2
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 2
- 230000036592 analgesia Effects 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 210000003484 anatomy Anatomy 0.000 description 2
- 230000003367 anti-collagen effect Effects 0.000 description 2
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 2
- 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 2
- 208000006673 asthma Diseases 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 201000000448 autoimmune hemolytic anemia Diseases 0.000 description 2
- 201000004982 autoimmune uveitis Diseases 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 2
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 125000004112 carboxyamino group Chemical group [H]OC(=O)N([H])[*] 0.000 description 2
- POIUWJQBRNEFGX-XAMSXPGMSA-N cathelicidin Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C1=CC=CC=C1 POIUWJQBRNEFGX-XAMSXPGMSA-N 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 102000027005 chemokine binding proteins Human genes 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 108010035886 connective tissue-activating peptide Proteins 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 2
- 229960000258 corticotropin Drugs 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000000430 cytokine receptor antagonist Substances 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 229920006237 degradable polymer Polymers 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 102100021145 fMet-Leu-Phe receptor Human genes 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 210000002683 foot Anatomy 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000011521 glass Substances 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
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 102000057593 human F8 Human genes 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 238000001802 infusion Methods 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
- 229960001388 interferon-beta Drugs 0.000 description 2
- 108090000237 interleukin-24 Proteins 0.000 description 2
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229940039781 leptin Drugs 0.000 description 2
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- VNYSSYRCGWBHLG-AMOLWHMGSA-M leukotriene B4(1-) Chemical compound CCCCC\C=C/C[C@@H](O)\C=C\C=C\C=C/[C@@H](O)CCCC([O-])=O VNYSSYRCGWBHLG-AMOLWHMGSA-M 0.000 description 2
- 150000002617 leukotrienes Chemical class 0.000 description 2
- 150000002639 lipoxins Chemical class 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 108010019677 lymphotactin Proteins 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 102000049853 macrophage stimulating protein Human genes 0.000 description 2
- 108010053292 macrophage stimulating protein Proteins 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- AEUKDPKXTPNBNY-XEYRWQBLSA-N mcp 2 Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)C1=CC=CC=C1 AEUKDPKXTPNBNY-XEYRWQBLSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000009126 molecular therapy Methods 0.000 description 2
- 206010028417 myasthenia gravis Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 239000003900 neurotrophic factor Substances 0.000 description 2
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 description 2
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 102000030769 platelet activating factor receptor Human genes 0.000 description 2
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 2
- 108010017992 platelet-derived growth factor C Proteins 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 208000005987 polymyositis Diseases 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000003405 preventing effect Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 229940097325 prolactin Drugs 0.000 description 2
- 208000005069 pulmonary fibrosis Diseases 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
- 229940047431 recombinate Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 201000000306 sarcoidosis Diseases 0.000 description 2
- 239000003001 serine protease inhibitor Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000007909 solid dosage form Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- WHNFPRLDDSXQCL-UAZQEYIDSA-N α-msh Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(N)=O)NC(=O)[C@H](CO)NC(C)=O)C1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UAZQEYIDSA-N 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- IESDGNYHXIOKRW-YXMSTPNBSA-N (2s)-2-[[(2s)-1-[(2s)-6-amino-2-[[(2s,3r)-2-amino-3-hydroxybutanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IESDGNYHXIOKRW-YXMSTPNBSA-N 0.000 description 1
- AXFMEGAFCUULFV-BLFANLJRSA-N (2s)-2-[[(2s)-1-[(2s,3r)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]pentanedioic acid Chemical compound CC[C@@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AXFMEGAFCUULFV-BLFANLJRSA-N 0.000 description 1
- NSZJXSMPGXGNJX-VWCSCAALSA-N (2s)-2-[[(2s)-2-[[(2s,3s)-2-[[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]propanoyl]amino]-3-methylpentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 NSZJXSMPGXGNJX-VWCSCAALSA-N 0.000 description 1
- DIBLBAURNYJYBF-XLXZRNDBSA-N (2s)-2-[[(2s)-2-[[2-[[(2s)-6-amino-2-[[(2s)-2-amino-3-methylbutanoyl]amino]hexanoyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@H](NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 DIBLBAURNYJYBF-XLXZRNDBSA-N 0.000 description 1
- WKVZMKDXJFCMMD-UVWUDEKDSA-L (5ar,8ar,9r)-5-[[(2r,4ar,6r,7r,8r,8as)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one;azanide;n,3-bis(2-chloroethyl)-2-ox Chemical compound [NH2-].[NH2-].Cl[Pt+2]Cl.ClCCNP1(=O)OCCCN1CCCl.COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3C(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 WKVZMKDXJFCMMD-UVWUDEKDSA-L 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- 102000007445 2',5'-Oligoadenylate Synthetase Human genes 0.000 description 1
- 108010086241 2',5'-Oligoadenylate Synthetase Proteins 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- UKPGFKQVRITNFM-KBPBESRZSA-N 2-[[2-[[(2s)-1-[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]acetic acid Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)NCC(O)=O)C1=CC=C(O)C=C1 UKPGFKQVRITNFM-KBPBESRZSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000228431 Acremonium chrysogenum Species 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 102000018918 Activin Receptors Human genes 0.000 description 1
- 108010052946 Activin Receptors Proteins 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 208000026872 Addison Disease Diseases 0.000 description 1
- 206010001497 Agitation Diseases 0.000 description 1
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 1
- DECCMEWNXSNSDO-ZLUOBGJFSA-N Ala-Cys-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O DECCMEWNXSNSDO-ZLUOBGJFSA-N 0.000 description 1
- JPGBXANAQYHTLA-DRZSPHRISA-N Ala-Gln-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JPGBXANAQYHTLA-DRZSPHRISA-N 0.000 description 1
- KXEVYGKATAMXJJ-ACZMJKKPSA-N Ala-Glu-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KXEVYGKATAMXJJ-ACZMJKKPSA-N 0.000 description 1
- VBRDBGCROKWTPV-XHNCKOQMSA-N Ala-Glu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N VBRDBGCROKWTPV-XHNCKOQMSA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 1
- DPNZTBKGAUAZQU-DLOVCJGASA-N Ala-Leu-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N DPNZTBKGAUAZQU-DLOVCJGASA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- OINVDEKBKBCPLX-JXUBOQSCSA-N Ala-Lys-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OINVDEKBKBCPLX-JXUBOQSCSA-N 0.000 description 1
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 1
- FEGOCLZUJUFCHP-CIUDSAMLSA-N Ala-Pro-Gln Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O FEGOCLZUJUFCHP-CIUDSAMLSA-N 0.000 description 1
- IORKCNUBHNIMKY-CIUDSAMLSA-N Ala-Pro-Glu Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O IORKCNUBHNIMKY-CIUDSAMLSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- YJHKTAMKPGFJCT-NRPADANISA-N Ala-Val-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O YJHKTAMKPGFJCT-NRPADANISA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 241000710929 Alphavirus Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 101100107610 Arabidopsis thaliana ABCF4 gene Proteins 0.000 description 1
- HJVGMOYJDDXLMI-AVGNSLFASA-N Arg-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N HJVGMOYJDDXLMI-AVGNSLFASA-N 0.000 description 1
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 1
- FLYANDHDFRGGTM-PYJNHQTQSA-N Arg-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FLYANDHDFRGGTM-PYJNHQTQSA-N 0.000 description 1
- BTJVOUQWFXABOI-IHRRRGAJSA-N Arg-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCNC(N)=N BTJVOUQWFXABOI-IHRRRGAJSA-N 0.000 description 1
- KMFPQTITXUKJOV-DCAQKATOSA-N Arg-Ser-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O KMFPQTITXUKJOV-DCAQKATOSA-N 0.000 description 1
- ICRHGPYYXMWHIE-LPEHRKFASA-N Arg-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ICRHGPYYXMWHIE-LPEHRKFASA-N 0.000 description 1
- ZJBUILVYSXQNSW-YTWAJWBKSA-N Arg-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O ZJBUILVYSXQNSW-YTWAJWBKSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- SLKLLQWZQHXYSV-CIUDSAMLSA-N Asn-Ala-Lys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O SLKLLQWZQHXYSV-CIUDSAMLSA-N 0.000 description 1
- BVLIJXXSXBUGEC-SRVKXCTJSA-N Asn-Asn-Tyr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O BVLIJXXSXBUGEC-SRVKXCTJSA-N 0.000 description 1
- WQLJRNRLHWJIRW-KKUMJFAQSA-N Asn-His-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)N)N)O WQLJRNRLHWJIRW-KKUMJFAQSA-N 0.000 description 1
- HDHZCEDPLTVHFZ-GUBZILKMSA-N Asn-Leu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O HDHZCEDPLTVHFZ-GUBZILKMSA-N 0.000 description 1
- RCFGLXMZDYNRSC-CIUDSAMLSA-N Asn-Lys-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O RCFGLXMZDYNRSC-CIUDSAMLSA-N 0.000 description 1
- ZUFPUBYQYWCMDB-NUMRIWBASA-N Asn-Thr-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZUFPUBYQYWCMDB-NUMRIWBASA-N 0.000 description 1
- LGCVSPFCFXWUEY-IHPCNDPISA-N Asn-Trp-Tyr Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)NC(=O)[C@H](CC(=O)N)N LGCVSPFCFXWUEY-IHPCNDPISA-N 0.000 description 1
- YQPSDMUGFKJZHR-QRTARXTBSA-N Asn-Trp-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)N)N YQPSDMUGFKJZHR-QRTARXTBSA-N 0.000 description 1
- QNNBHTFDFFFHGC-KKUMJFAQSA-N Asn-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QNNBHTFDFFFHGC-KKUMJFAQSA-N 0.000 description 1
- MYRLSKYSMXNLLA-LAEOZQHASA-N Asn-Val-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MYRLSKYSMXNLLA-LAEOZQHASA-N 0.000 description 1
- KBQOUDLMWYWXNP-YDHLFZDLSA-N Asn-Val-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CC(=O)N)N KBQOUDLMWYWXNP-YDHLFZDLSA-N 0.000 description 1
- LKIYSIYBKYLKPU-BIIVOSGPSA-N Asp-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O LKIYSIYBKYLKPU-BIIVOSGPSA-N 0.000 description 1
- AMRANMVXQWXNAH-ZLUOBGJFSA-N Asp-Cys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC(O)=O AMRANMVXQWXNAH-ZLUOBGJFSA-N 0.000 description 1
- HRGGPWBIMIQANI-GUBZILKMSA-N Asp-Gln-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HRGGPWBIMIQANI-GUBZILKMSA-N 0.000 description 1
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 1
- WSGVTKZFVJSJOG-RCOVLWMOSA-N Asp-Gly-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O WSGVTKZFVJSJOG-RCOVLWMOSA-N 0.000 description 1
- LIQNMKIBMPEOOP-IHRRRGAJSA-N Asp-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC(=O)O)N LIQNMKIBMPEOOP-IHRRRGAJSA-N 0.000 description 1
- HJZLUGQGJWXJCJ-CIUDSAMLSA-N Asp-Pro-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O HJZLUGQGJWXJCJ-CIUDSAMLSA-N 0.000 description 1
- AHWRSSLYSGLBGD-CIUDSAMLSA-N Asp-Pro-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AHWRSSLYSGLBGD-CIUDSAMLSA-N 0.000 description 1
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 1
- YUELDQUPTAYEGM-XIRDDKMYSA-N Asp-Trp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CC(=O)O)N YUELDQUPTAYEGM-XIRDDKMYSA-N 0.000 description 1
- FIRWLDUOFOULCA-XIRDDKMYSA-N Asp-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N FIRWLDUOFOULCA-XIRDDKMYSA-N 0.000 description 1
- PLNJUJGNLDSFOP-UWJYBYFXSA-N Asp-Tyr-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PLNJUJGNLDSFOP-UWJYBYFXSA-N 0.000 description 1
- MFDPBZAFCRKYEY-LAEOZQHASA-N Asp-Val-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MFDPBZAFCRKYEY-LAEOZQHASA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241001367049 Autographa Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 241000212384 Bifora Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000282817 Bovidae Species 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108700012434 CCL3 Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000034628 Celiac artery compression syndrome Diseases 0.000 description 1
- 102000000013 Chemokine CCL3 Human genes 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 102000002734 Collagen Type VI Human genes 0.000 description 1
- 108010043741 Collagen Type VI Proteins 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- HRJLVSQKBLZHSR-ZLUOBGJFSA-N Cys-Asn-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O HRJLVSQKBLZHSR-ZLUOBGJFSA-N 0.000 description 1
- XIZWKXATMJODQW-KKUMJFAQSA-N Cys-His-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CS)N XIZWKXATMJODQW-KKUMJFAQSA-N 0.000 description 1
- YFAFBAPQHGULQT-HJPIBITLSA-N Cys-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CS)N YFAFBAPQHGULQT-HJPIBITLSA-N 0.000 description 1
- OHLLDUNVMPPUMD-DCAQKATOSA-N Cys-Leu-Val Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N OHLLDUNVMPPUMD-DCAQKATOSA-N 0.000 description 1
- VDUPGIDTWNQAJD-CIUDSAMLSA-N Cys-Lys-Cys Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CS)C(O)=O VDUPGIDTWNQAJD-CIUDSAMLSA-N 0.000 description 1
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 1
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 1
- HJXSYJVCMUOUNY-SRVKXCTJSA-N Cys-Ser-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N HJXSYJVCMUOUNY-SRVKXCTJSA-N 0.000 description 1
- NDNZRWUDUMTITL-FXQIFTODSA-N Cys-Ser-Val Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NDNZRWUDUMTITL-FXQIFTODSA-N 0.000 description 1
- ALTQTAKGRFLRLR-GUBZILKMSA-N Cys-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CS)N ALTQTAKGRFLRLR-GUBZILKMSA-N 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 238000011763 DBA/1J (JAX™ mouse strain) Methods 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010064769 Dactylitis Diseases 0.000 description 1
- 101000937129 Drosophila melanogaster Cadherin-related tumor suppressor Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010049140 Endorphins Proteins 0.000 description 1
- 102000009025 Endorphins Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 206010015943 Eye inflammation Diseases 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- RZSLYUUFFVHFRQ-FXQIFTODSA-N Gln-Ala-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O RZSLYUUFFVHFRQ-FXQIFTODSA-N 0.000 description 1
- NVEASDQHBRZPSU-BQBZGAKWSA-N Gln-Gln-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O NVEASDQHBRZPSU-BQBZGAKWSA-N 0.000 description 1
- SNLOOPZHAQDMJG-CIUDSAMLSA-N Gln-Glu-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SNLOOPZHAQDMJG-CIUDSAMLSA-N 0.000 description 1
- HMIXCETWRYDVMO-GUBZILKMSA-N Gln-Pro-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O HMIXCETWRYDVMO-GUBZILKMSA-N 0.000 description 1
- PAOHIZNRJNIXQY-XQXXSGGOSA-N Gln-Thr-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PAOHIZNRJNIXQY-XQXXSGGOSA-N 0.000 description 1
- CSMHMEATMDCQNY-DZKIICNBSA-N Gln-Val-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CSMHMEATMDCQNY-DZKIICNBSA-N 0.000 description 1
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- CVPXINNKRTZBMO-CIUDSAMLSA-N Glu-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N CVPXINNKRTZBMO-CIUDSAMLSA-N 0.000 description 1
- SRZLHYPAOXBBSB-HJGDQZAQSA-N Glu-Arg-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SRZLHYPAOXBBSB-HJGDQZAQSA-N 0.000 description 1
- GLWXKFRTOHKGIT-ACZMJKKPSA-N Glu-Asn-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O GLWXKFRTOHKGIT-ACZMJKKPSA-N 0.000 description 1
- CYHBMLHCQXXCCT-AVGNSLFASA-N Glu-Asp-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O CYHBMLHCQXXCCT-AVGNSLFASA-N 0.000 description 1
- XHWLNISLUFEWNS-CIUDSAMLSA-N Glu-Gln-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O XHWLNISLUFEWNS-CIUDSAMLSA-N 0.000 description 1
- HUFCEIHAFNVSNR-IHRRRGAJSA-N Glu-Gln-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HUFCEIHAFNVSNR-IHRRRGAJSA-N 0.000 description 1
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- NWOUBJNMZDDGDT-AVGNSLFASA-N Glu-Leu-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 NWOUBJNMZDDGDT-AVGNSLFASA-N 0.000 description 1
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 1
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 description 1
- AAJHGGDRKHYSDH-GUBZILKMSA-N Glu-Pro-Gln Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O AAJHGGDRKHYSDH-GUBZILKMSA-N 0.000 description 1
- ALMBZBOCGSVSAI-ACZMJKKPSA-N Glu-Ser-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ALMBZBOCGSVSAI-ACZMJKKPSA-N 0.000 description 1
- RFTVTKBHDXCEEX-WDSKDSINSA-N Glu-Ser-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RFTVTKBHDXCEEX-WDSKDSINSA-N 0.000 description 1
- CAQXJMUDOLSBPF-SUSMZKCASA-N Glu-Thr-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAQXJMUDOLSBPF-SUSMZKCASA-N 0.000 description 1
- MFYLRRCYBBJYPI-JYJNAYRXSA-N Glu-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O MFYLRRCYBBJYPI-JYJNAYRXSA-N 0.000 description 1
- WGYHAAXZWPEBDQ-IFFSRLJSSA-N Glu-Val-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGYHAAXZWPEBDQ-IFFSRLJSSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- DTPOVRRYXPJJAZ-FJXKBIBVSA-N Gly-Arg-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N DTPOVRRYXPJJAZ-FJXKBIBVSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-N 0.000 description 1
- FZQLXNIMCPJVJE-YUMQZZPRSA-N Gly-Asp-Leu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O FZQLXNIMCPJVJE-YUMQZZPRSA-N 0.000 description 1
- PABFFPWEJMEVEC-JGVFFNPUSA-N Gly-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)CN)C(=O)O PABFFPWEJMEVEC-JGVFFNPUSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- YIFUFYZELCMPJP-YUMQZZPRSA-N Gly-Leu-Cys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O YIFUFYZELCMPJP-YUMQZZPRSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 1
- JSLVAHYTAJJEQH-QWRGUYRKSA-N Gly-Ser-Phe Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 JSLVAHYTAJJEQH-QWRGUYRKSA-N 0.000 description 1
- RIUZKUJUPVFAGY-HOTGVXAUSA-N Gly-Trp-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)NC(=O)CN RIUZKUJUPVFAGY-HOTGVXAUSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- WMKXFMUJRCEGRP-SRVKXCTJSA-N His-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N WMKXFMUJRCEGRP-SRVKXCTJSA-N 0.000 description 1
- AKEDPWJFQULLPE-IUCAKERBSA-N His-Glu-Gly Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O AKEDPWJFQULLPE-IUCAKERBSA-N 0.000 description 1
- CNHSMSFYVARZLI-YJRXYDGGSA-N His-His-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CNHSMSFYVARZLI-YJRXYDGGSA-N 0.000 description 1
- LVXFNTIIGOQBMD-SRVKXCTJSA-N His-Leu-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O LVXFNTIIGOQBMD-SRVKXCTJSA-N 0.000 description 1
- HYWZHNUGAYVEEW-KKUMJFAQSA-N His-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N HYWZHNUGAYVEEW-KKUMJFAQSA-N 0.000 description 1
- CUEQQFOGARVNHU-VGDYDELISA-N His-Ser-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CUEQQFOGARVNHU-VGDYDELISA-N 0.000 description 1
- BRQKGRLDDDQWQJ-MBLNEYKQSA-N His-Thr-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O BRQKGRLDDDQWQJ-MBLNEYKQSA-N 0.000 description 1
- DEMIXZCKUXVEBO-BWAGICSOSA-N His-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N)O DEMIXZCKUXVEBO-BWAGICSOSA-N 0.000 description 1
- 101000883515 Homo sapiens Chitinase-3-like protein 1 Proteins 0.000 description 1
- 101001044895 Homo sapiens Interleukin-20 receptor subunit beta Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- UNDGQKWQNSTPPW-CYDGBPFRSA-N Ile-Arg-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCSC)C(=O)O)N UNDGQKWQNSTPPW-CYDGBPFRSA-N 0.000 description 1
- GYAFMRQGWHXMII-IUKAMOBKSA-N Ile-Asp-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N GYAFMRQGWHXMII-IUKAMOBKSA-N 0.000 description 1
- ZGGWRNBSBOHIGH-HVTMNAMFSA-N Ile-Gln-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ZGGWRNBSBOHIGH-HVTMNAMFSA-N 0.000 description 1
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-N 0.000 description 1
- RMNMUUCYTMLWNA-ZPFDUUQYSA-N Ile-Lys-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N RMNMUUCYTMLWNA-ZPFDUUQYSA-N 0.000 description 1
- GLYJPWIRLBAIJH-FQUUOJAGSA-N Ile-Lys-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N GLYJPWIRLBAIJH-FQUUOJAGSA-N 0.000 description 1
- GLYJPWIRLBAIJH-UHFFFAOYSA-N Ile-Lys-Pro Natural products CCC(C)C(N)C(=O)NC(CCCCN)C(=O)N1CCCC1C(O)=O GLYJPWIRLBAIJH-UHFFFAOYSA-N 0.000 description 1
- BBIXOODYWPFNDT-CIUDSAMLSA-N Ile-Pro Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O BBIXOODYWPFNDT-CIUDSAMLSA-N 0.000 description 1
- PELCGFMHLZXWBQ-BJDJZHNGSA-N Ile-Ser-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)O)N PELCGFMHLZXWBQ-BJDJZHNGSA-N 0.000 description 1
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 1
- JDCQDJVYUXNCGF-SPOWBLRKSA-N Ile-Ser-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N JDCQDJVYUXNCGF-SPOWBLRKSA-N 0.000 description 1
- ZUWSVOYKBCHLRR-MGHWNKPDSA-N Ile-Tyr-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N ZUWSVOYKBCHLRR-MGHWNKPDSA-N 0.000 description 1
- WRDTXMBPHMBGIB-STECZYCISA-N Ile-Tyr-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=C(O)C=C1 WRDTXMBPHMBGIB-STECZYCISA-N 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 102100026818 Inhibin beta E chain Human genes 0.000 description 1
- 108010004250 Inhibins Proteins 0.000 description 1
- 102000002746 Inhibins Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 102100022339 Integrin alpha-L Human genes 0.000 description 1
- 102100035018 Interleukin-17 receptor A Human genes 0.000 description 1
- 101710186083 Interleukin-17 receptor A Proteins 0.000 description 1
- 101710186071 Interleukin-17 receptor B Proteins 0.000 description 1
- 102100035014 Interleukin-17 receptor B Human genes 0.000 description 1
- 102100035012 Interleukin-17 receptor C Human genes 0.000 description 1
- 101710186068 Interleukin-17 receptor C Proteins 0.000 description 1
- 102100035015 Interleukin-17 receptor D Human genes 0.000 description 1
- 101710186062 Interleukin-17 receptor D Proteins 0.000 description 1
- 102100035016 Interleukin-17 receptor E Human genes 0.000 description 1
- 101710186076 Interleukin-17 receptor E Proteins 0.000 description 1
- 102100022705 Interleukin-20 receptor subunit beta Human genes 0.000 description 1
- 102100040066 Interleukin-27 receptor subunit alpha Human genes 0.000 description 1
- 101710089672 Interleukin-27 receptor subunit alpha Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- LJHGALIOHLRRQN-DCAQKATOSA-N Leu-Ala-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N LJHGALIOHLRRQN-DCAQKATOSA-N 0.000 description 1
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 1
- HYIFFZAQXPUEAU-QWRGUYRKSA-N Leu-Gly-Leu Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C HYIFFZAQXPUEAU-QWRGUYRKSA-N 0.000 description 1
- BTNXKBVLWJBTNR-SRVKXCTJSA-N Leu-His-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(O)=O BTNXKBVLWJBTNR-SRVKXCTJSA-N 0.000 description 1
- BKTXKJMNTSMJDQ-AVGNSLFASA-N Leu-His-Gln Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N BKTXKJMNTSMJDQ-AVGNSLFASA-N 0.000 description 1
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 1
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 1
- CHJKEDSZNSONPS-DCAQKATOSA-N Leu-Pro-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O CHJKEDSZNSONPS-DCAQKATOSA-N 0.000 description 1
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 1
- LINKCQUOMUDLKN-KATARQTJSA-N Leu-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(C)C)N)O LINKCQUOMUDLKN-KATARQTJSA-N 0.000 description 1
- 102100021747 Leukemia inhibitory factor receptor Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 1
- 206010062049 Lymphocytic infiltration Diseases 0.000 description 1
- KCXUCYYZNZFGLL-SRVKXCTJSA-N Lys-Ala-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O KCXUCYYZNZFGLL-SRVKXCTJSA-N 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- YIBOAHAOAWACDK-QEJZJMRPSA-N Lys-Ala-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YIBOAHAOAWACDK-QEJZJMRPSA-N 0.000 description 1
- ALSRJRIWBNENFY-DCAQKATOSA-N Lys-Arg-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O ALSRJRIWBNENFY-DCAQKATOSA-N 0.000 description 1
- YKIRNDPUWONXQN-GUBZILKMSA-N Lys-Asn-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N YKIRNDPUWONXQN-GUBZILKMSA-N 0.000 description 1
- FLCMXEFCTLXBTL-DCAQKATOSA-N Lys-Asp-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N FLCMXEFCTLXBTL-DCAQKATOSA-N 0.000 description 1
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 1
- KWUKZRFFKPLUPE-HJGDQZAQSA-N Lys-Asp-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWUKZRFFKPLUPE-HJGDQZAQSA-N 0.000 description 1
- NDORZBUHCOJQDO-GVXVVHGQSA-N Lys-Gln-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O NDORZBUHCOJQDO-GVXVVHGQSA-N 0.000 description 1
- XNKDCYABMBBEKN-IUCAKERBSA-N Lys-Gly-Gln Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O XNKDCYABMBBEKN-IUCAKERBSA-N 0.000 description 1
- YDDDRTIPNTWGIG-SRVKXCTJSA-N Lys-Lys-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O YDDDRTIPNTWGIG-SRVKXCTJSA-N 0.000 description 1
- ODTZHNZPINULEU-KKUMJFAQSA-N Lys-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCCN)N ODTZHNZPINULEU-KKUMJFAQSA-N 0.000 description 1
- WQDKIVRHTQYJSN-DCAQKATOSA-N Lys-Ser-Arg Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WQDKIVRHTQYJSN-DCAQKATOSA-N 0.000 description 1
- IOQWIOPSKJOEKI-SRVKXCTJSA-N Lys-Ser-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O IOQWIOPSKJOEKI-SRVKXCTJSA-N 0.000 description 1
- IEVXCWPVBYCJRZ-IXOXFDKPSA-N Lys-Thr-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IEVXCWPVBYCJRZ-IXOXFDKPSA-N 0.000 description 1
- DLCAXBGXGOVUCD-PPCPHDFISA-N Lys-Thr-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DLCAXBGXGOVUCD-PPCPHDFISA-N 0.000 description 1
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 1
- GILLQRYAWOMHED-DCAQKATOSA-N Lys-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN GILLQRYAWOMHED-DCAQKATOSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- IVCPHARVJUYDPA-FXQIFTODSA-N Met-Asn-Asp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N IVCPHARVJUYDPA-FXQIFTODSA-N 0.000 description 1
- LXCSZPUQKMTXNW-BQBZGAKWSA-N Met-Ser-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O LXCSZPUQKMTXNW-BQBZGAKWSA-N 0.000 description 1
- IHRFZLQEQVHXFA-RHYQMDGZSA-N Met-Thr-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCCN IHRFZLQEQVHXFA-RHYQMDGZSA-N 0.000 description 1
- IIHMNTBFPMRJCN-RCWTZXSCSA-N Met-Val-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IIHMNTBFPMRJCN-RCWTZXSCSA-N 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 101710151805 Mitochondrial intermediate peptidase 1 Proteins 0.000 description 1
- 101100369641 Mus musculus Tigit gene Proteins 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 241000713883 Myeloproliferative sarcoma virus Species 0.000 description 1
- 241001477931 Mythimna unipuncta Species 0.000 description 1
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102100029268 Neurotrophin-3 Human genes 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 108010064527 OSM-LIF Receptors Proteins 0.000 description 1
- 102000003840 Opioid Receptors Human genes 0.000 description 1
- 108090000137 Opioid Receptors Proteins 0.000 description 1
- 239000008896 Opium Substances 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 201000011152 Pemphigus Diseases 0.000 description 1
- 241000721454 Pemphigus Species 0.000 description 1
- CPTJPDZTFNKFOU-MXAVVETBSA-N Phe-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N CPTJPDZTFNKFOU-MXAVVETBSA-N 0.000 description 1
- VZFPYFRVHMSSNA-JURCDPSOSA-N Phe-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=CC=C1 VZFPYFRVHMSSNA-JURCDPSOSA-N 0.000 description 1
- KDYPMIZMXDECSU-JYJNAYRXSA-N Phe-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 KDYPMIZMXDECSU-JYJNAYRXSA-N 0.000 description 1
- LRBSWBVUCLLRLU-BZSNNMDCSA-N Phe-Leu-Lys Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(O)=O LRBSWBVUCLLRLU-BZSNNMDCSA-N 0.000 description 1
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 1
- WEDZFLRYSIDIRX-IHRRRGAJSA-N Phe-Ser-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=CC=C1 WEDZFLRYSIDIRX-IHRRRGAJSA-N 0.000 description 1
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 1
- IIEOLPMQYRBZCN-SRVKXCTJSA-N Phe-Ser-Cys Chemical compound N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O IIEOLPMQYRBZCN-SRVKXCTJSA-N 0.000 description 1
- SJRQWEDYTKYHHL-SLFFLAALSA-N Phe-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O SJRQWEDYTKYHHL-SLFFLAALSA-N 0.000 description 1
- GOUWCZRDTWTODO-YDHLFZDLSA-N Phe-Val-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O GOUWCZRDTWTODO-YDHLFZDLSA-N 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 1
- TUYWCHPXKQTISF-LPEHRKFASA-N Pro-Cys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CS)C(=O)N2CCC[C@@H]2C(=O)O TUYWCHPXKQTISF-LPEHRKFASA-N 0.000 description 1
- UPJGUQPLYWTISV-GUBZILKMSA-N Pro-Gln-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UPJGUQPLYWTISV-GUBZILKMSA-N 0.000 description 1
- LHALYDBUDCWMDY-CIUDSAMLSA-N Pro-Glu-Ala Chemical compound C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1)C(O)=O LHALYDBUDCWMDY-CIUDSAMLSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- LGSANCBHSMDFDY-GARJFASQSA-N Pro-Glu-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N2CCC[C@@H]2C(=O)O LGSANCBHSMDFDY-GARJFASQSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 1
- ZLXKLMHAMDENIO-DCAQKATOSA-N Pro-Lys-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLXKLMHAMDENIO-DCAQKATOSA-N 0.000 description 1
- ABSSTGUCBCDKMU-UWVGGRQHSA-N Pro-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 ABSSTGUCBCDKMU-UWVGGRQHSA-N 0.000 description 1
- ULWBBFKQBDNGOY-RWMBFGLXSA-N Pro-Lys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N2CCC[C@@H]2C(=O)O ULWBBFKQBDNGOY-RWMBFGLXSA-N 0.000 description 1
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 1
- LEIKGVHQTKHOLM-IUCAKERBSA-N Pro-Pro-Gly Chemical compound OC(=O)CNC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 LEIKGVHQTKHOLM-IUCAKERBSA-N 0.000 description 1
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 description 1
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- QKDIHFHGHBYTKB-IHRRRGAJSA-N Pro-Ser-Phe Chemical compound N([C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1 QKDIHFHGHBYTKB-IHRRRGAJSA-N 0.000 description 1
- PRKWBYCXBBSLSK-GUBZILKMSA-N Pro-Ser-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O PRKWBYCXBBSLSK-GUBZILKMSA-N 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- UEJYSALTSUZXFV-SRVKXCTJSA-N Rigin Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O UEJYSALTSUZXFV-SRVKXCTJSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101100068078 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GCN4 gene Proteins 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- SRTCFKGBYBZRHA-ACZMJKKPSA-N Ser-Ala-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SRTCFKGBYBZRHA-ACZMJKKPSA-N 0.000 description 1
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 1
- QEDMOZUJTGEIBF-FXQIFTODSA-N Ser-Arg-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O QEDMOZUJTGEIBF-FXQIFTODSA-N 0.000 description 1
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 1
- HZWAHWQZPSXNCB-BPUTZDHNSA-N Ser-Arg-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O HZWAHWQZPSXNCB-BPUTZDHNSA-N 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- VGNYHOBZJKWRGI-CIUDSAMLSA-N Ser-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CO VGNYHOBZJKWRGI-CIUDSAMLSA-N 0.000 description 1
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 1
- BYIROAKULFFTEK-CIUDSAMLSA-N Ser-Asp-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO BYIROAKULFFTEK-CIUDSAMLSA-N 0.000 description 1
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-N 0.000 description 1
- SWIQQMYVHIXPEK-FXQIFTODSA-N Ser-Cys-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O SWIQQMYVHIXPEK-FXQIFTODSA-N 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 1
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 1
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 1
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 1
- VVKVHAOOUGNDPJ-SRVKXCTJSA-N Ser-Tyr-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VVKVHAOOUGNDPJ-SRVKXCTJSA-N 0.000 description 1
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 1
- IAOHCSQDQDWRQU-GUBZILKMSA-N Ser-Val-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IAOHCSQDQDWRQU-GUBZILKMSA-N 0.000 description 1
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 1
- HNDMFDBQXYZSRM-IHRRRGAJSA-N Ser-Val-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HNDMFDBQXYZSRM-IHRRRGAJSA-N 0.000 description 1
- 241000710960 Sindbis virus Species 0.000 description 1
- 241000256247 Spodoptera exigua Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 206010042742 Sympathetic ophthalmia Diseases 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- LVHHEVGYAZGXDE-KDXUFGMBSA-N Thr-Ala-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(=O)O)N)O LVHHEVGYAZGXDE-KDXUFGMBSA-N 0.000 description 1
- ASJDFGOPDCVXTG-KATARQTJSA-N Thr-Cys-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O ASJDFGOPDCVXTG-KATARQTJSA-N 0.000 description 1
- KWQBJOUOSNJDRR-XAVMHZPKSA-N Thr-Cys-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N)O KWQBJOUOSNJDRR-XAVMHZPKSA-N 0.000 description 1
- DSLHSTIUAPKERR-XGEHTFHBSA-N Thr-Cys-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O DSLHSTIUAPKERR-XGEHTFHBSA-N 0.000 description 1
- LAFLAXHTDVNVEL-WDCWCFNPSA-N Thr-Gln-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O LAFLAXHTDVNVEL-WDCWCFNPSA-N 0.000 description 1
- LHEZGZQRLDBSRR-WDCWCFNPSA-N Thr-Glu-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LHEZGZQRLDBSRR-WDCWCFNPSA-N 0.000 description 1
- XOTBWOCSLMBGMF-SUSMZKCASA-N Thr-Glu-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XOTBWOCSLMBGMF-SUSMZKCASA-N 0.000 description 1
- XFTYVCHLARBHBQ-FOHZUACHSA-N Thr-Gly-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XFTYVCHLARBHBQ-FOHZUACHSA-N 0.000 description 1
- NQVDGKYAUHTCME-QTKMDUPCSA-N Thr-His-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O NQVDGKYAUHTCME-QTKMDUPCSA-N 0.000 description 1
- SXAGUVRFGJSFKC-ZEILLAHLSA-N Thr-His-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SXAGUVRFGJSFKC-ZEILLAHLSA-N 0.000 description 1
- CRZNCABIJLRFKZ-IUKAMOBKSA-N Thr-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N CRZNCABIJLRFKZ-IUKAMOBKSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 1
- TZJSEJOXAIWOST-RHYQMDGZSA-N Thr-Lys-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N TZJSEJOXAIWOST-RHYQMDGZSA-N 0.000 description 1
- BDGBHYCAZJPLHX-HJGDQZAQSA-N Thr-Lys-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BDGBHYCAZJPLHX-HJGDQZAQSA-N 0.000 description 1
- XSEPSRUDSPHMPX-KATARQTJSA-N Thr-Lys-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O XSEPSRUDSPHMPX-KATARQTJSA-N 0.000 description 1
- PZSDPRBZINDEJV-HTUGSXCWSA-N Thr-Phe-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O PZSDPRBZINDEJV-HTUGSXCWSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- BEZTUFWTPVOROW-KJEVXHAQSA-N Thr-Tyr-Arg Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N)O BEZTUFWTPVOROW-KJEVXHAQSA-N 0.000 description 1
- ABCLYRRGTZNIFU-BWAGICSOSA-N Thr-Tyr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N)O ABCLYRRGTZNIFU-BWAGICSOSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 208000024799 Thyroid disease Diseases 0.000 description 1
- ADBFWLXCCKIXBQ-XIRDDKMYSA-N Trp-Asn-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N ADBFWLXCCKIXBQ-XIRDDKMYSA-N 0.000 description 1
- DQDXHYIEITXNJY-BPUTZDHNSA-N Trp-Gln-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N DQDXHYIEITXNJY-BPUTZDHNSA-N 0.000 description 1
- PHNBFZBKLWEBJN-BPUTZDHNSA-N Trp-Glu-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PHNBFZBKLWEBJN-BPUTZDHNSA-N 0.000 description 1
- HNIWONZFMIPCCT-SIXJUCDHSA-N Trp-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N HNIWONZFMIPCCT-SIXJUCDHSA-N 0.000 description 1
- UPNRACRNHISCAF-SZMVWBNQSA-N Trp-Lys-Gln Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O)=CNC2=C1 UPNRACRNHISCAF-SZMVWBNQSA-N 0.000 description 1
- GEGYPBOPIGNZIF-CWRNSKLLSA-N Trp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N)C(=O)O GEGYPBOPIGNZIF-CWRNSKLLSA-N 0.000 description 1
- HHPSUFUXXBOFQY-AQZXSJQPSA-N Trp-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O HHPSUFUXXBOFQY-AQZXSJQPSA-N 0.000 description 1
- SSSDKJMQMZTMJP-BVSLBCMMSA-N Trp-Tyr-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CC=C(O)C=C1 SSSDKJMQMZTMJP-BVSLBCMMSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- QYSBJAUCUKHSLU-JYJNAYRXSA-N Tyr-Arg-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O QYSBJAUCUKHSLU-JYJNAYRXSA-N 0.000 description 1
- SCCKSNREWHMKOJ-SRVKXCTJSA-N Tyr-Asn-Ser Chemical compound N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O SCCKSNREWHMKOJ-SRVKXCTJSA-N 0.000 description 1
- CVXURBLRELTJKO-BWAGICSOSA-N Tyr-His-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)O CVXURBLRELTJKO-BWAGICSOSA-N 0.000 description 1
- GZUIDWDVMWZSMI-KKUMJFAQSA-N Tyr-Lys-Cys Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CS)C(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GZUIDWDVMWZSMI-KKUMJFAQSA-N 0.000 description 1
- SINRIKQYQJRGDQ-MEYUZBJRSA-N Tyr-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 SINRIKQYQJRGDQ-MEYUZBJRSA-N 0.000 description 1
- BGFCXQXETBDEHP-BZSNNMDCSA-N Tyr-Phe-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O BGFCXQXETBDEHP-BZSNNMDCSA-N 0.000 description 1
- WPRVVBVWIUWLOH-UFYCRDLUSA-N Tyr-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CC2=CC=C(C=C2)O)N WPRVVBVWIUWLOH-UFYCRDLUSA-N 0.000 description 1
- RIVVDNTUSRVTQT-IRIUXVKKSA-N Tyr-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O RIVVDNTUSRVTQT-IRIUXVKKSA-N 0.000 description 1
- ZZDYJFVIKVSUFA-WLTAIBSBSA-N Tyr-Thr-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O ZZDYJFVIKVSUFA-WLTAIBSBSA-N 0.000 description 1
- HZWPGKAKGYJWCI-ULQDDVLXSA-N Tyr-Val-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(C)C)C(O)=O HZWPGKAKGYJWCI-ULQDDVLXSA-N 0.000 description 1
- 244000301083 Ustilago maydis Species 0.000 description 1
- 235000015919 Ustilago maydis Nutrition 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- BYOHPUZJVXWHAE-BYULHYEWSA-N Val-Asn-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N BYOHPUZJVXWHAE-BYULHYEWSA-N 0.000 description 1
- QHDXUYOYTPWCSK-RCOVLWMOSA-N Val-Asp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)O)N QHDXUYOYTPWCSK-RCOVLWMOSA-N 0.000 description 1
- TZVUSFMQWPWHON-NHCYSSNCSA-N Val-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N TZVUSFMQWPWHON-NHCYSSNCSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- QHFQQRKNGCXTHL-AUTRQRHGSA-N Val-Gln-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QHFQQRKNGCXTHL-AUTRQRHGSA-N 0.000 description 1
- RKIGNDAHUOOIMJ-BQFCYCMXSA-N Val-Glu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)C(C)C)C(O)=O)=CNC2=C1 RKIGNDAHUOOIMJ-BQFCYCMXSA-N 0.000 description 1
- LYERIXUFCYVFFX-GVXVVHGQSA-N Val-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N LYERIXUFCYVFFX-GVXVVHGQSA-N 0.000 description 1
- XTDDIVQWDXMRJL-IHRRRGAJSA-N Val-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N XTDDIVQWDXMRJL-IHRRRGAJSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- HPANGHISDXDUQY-ULQDDVLXSA-N Val-Lys-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HPANGHISDXDUQY-ULQDDVLXSA-N 0.000 description 1
- FMQGYTMERWBMSI-HJWJTTGWSA-N Val-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N FMQGYTMERWBMSI-HJWJTTGWSA-N 0.000 description 1
- VIKZGAUAKQZDOF-NRPADANISA-N Val-Ser-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O VIKZGAUAKQZDOF-NRPADANISA-N 0.000 description 1
- QTPQHINADBYBNA-DCAQKATOSA-N Val-Ser-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN QTPQHINADBYBNA-DCAQKATOSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- BZDGLJPROOOUOZ-XGEHTFHBSA-N Val-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N)O BZDGLJPROOOUOZ-XGEHTFHBSA-N 0.000 description 1
- BGTDGENDNWGMDQ-KJEVXHAQSA-N Val-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](C(C)C)N)O BGTDGENDNWGMDQ-KJEVXHAQSA-N 0.000 description 1
- JSOXWWFKRJKTMT-WOPDTQHZSA-N Val-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N JSOXWWFKRJKTMT-WOPDTQHZSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- AZYNOAMYXWYHSQ-UHFFFAOYSA-N [NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[O-]P([O-])([O-])=O.[O-]S([O-])(=O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O Chemical compound [NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[NH4+].[O-]P([O-])([O-])=O.[O-]S([O-])(=O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O AZYNOAMYXWYHSQ-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000007801 affinity label Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010041407 alanylaspartic acid Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 208000004631 alopecia areata Diseases 0.000 description 1
- 229940087168 alpha tocopherol Drugs 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010013835 arginine glutamate Proteins 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 230000002917 arthritic effect Effects 0.000 description 1
- 210000000544 articulatio talocruralis Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000005311 autocorrelation function Methods 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- 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 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- BBWBEZAMXFGUGK-UHFFFAOYSA-N bis(dodecylsulfanyl)-methylarsane Chemical compound CCCCCCCCCCCCS[As](C)SCCCCCCCCCCCC BBWBEZAMXFGUGK-UHFFFAOYSA-N 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000000139 costimulatory 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
- 238000004132 cross linking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 238000007046 ethoxylation reaction Methods 0.000 description 1
- BFMKFCLXZSUVPI-UHFFFAOYSA-N ethyl but-3-enoate Chemical compound CCOC(=O)CC=C BFMKFCLXZSUVPI-UHFFFAOYSA-N 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012537 formulation buffer Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 230000018981 granulocyte chemotaxis Effects 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- 210000002837 heart atrium Anatomy 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 102000054350 human CHI3L1 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000037903 inflammatory enteropathy Diseases 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 102000048260 kappa Opioid Receptors Human genes 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 238000000670 ligand binding assay Methods 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- IXAQOQZEOGMIQS-SSQFXEBMSA-N lipoxin A4 Chemical compound CCCCC[C@H](O)\C=C\C=C/C=C/C=C/[C@@H](O)[C@@H](O)CCCC(O)=O IXAQOQZEOGMIQS-SSQFXEBMSA-N 0.000 description 1
- 239000008263 liquid aerosol Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000012516 mab select resin Substances 0.000 description 1
- 201000006512 mast cell neoplasm Diseases 0.000 description 1
- 208000006971 mastocytoma Diseases 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 238000000569 multi-angle light scattering Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 229960001027 opium Drugs 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 201000001976 pemphigus vulgaris Diseases 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 238000013423 psoriatic mouse model Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 239000010458 rotten stone Substances 0.000 description 1
- 102200078510 rs121964962 Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229920002545 silicone oil Polymers 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010129 solution processing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002889 sympathetic effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 1
- 208000021510 thyroid gland disease Diseases 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
- 210000003857 wrist joint Anatomy 0.000 description 1
- 108010082737 zymolyase Proteins 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
- 108020001588 κ-opioid receptors Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Rheumatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurology (AREA)
- Pain & Pain Management (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Physical Education & Sports Medicine (AREA)
- Neurosurgery (AREA)
- Biomedical Technology (AREA)
- Transplantation (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
公开了与可溶性二聚蛋白有关的组合物和方法。所述二聚蛋白包含经由二聚化结构域连接的第一种和第二种多肽融合体,每种多肽融合体包含与细胞因子或细胞表面受体的细胞外结构域相对应的第一个和第二个单体结构域。所述单体结构域可以位于所述二聚化结构域的氨基端和羧基端。可替换地,所述单体结构域可以串联地位于所述二聚化结构域的羧基端或氨基端。所述二聚蛋白在用于治疗、诊断和研究的方法中是有用的。
Description
相关申请的交叉引用
本申请要求2010年6月9日提交的美国临时申请系列号61/352,873的权益。
发明背景
细胞表面受体通过结合它们的相应配体来介导多种生物学效应。受体通常由一种或多种嵌膜蛋白组成,所述嵌膜蛋白以高亲和力结合配体(诸如细胞因子或反受体),并通过某些受体亚基的细胞质部分将该结合事件转导给细胞。基于它们的细胞外配体结合结构域的相似性,已经将细胞表面受体分成几类。
共刺激性受体在介导免疫细胞功能中的作用,例证了细胞表面受体的生理重要性。MHC分子和由抗原呈递细胞(APC)呈递的外来肽与T细胞抗原受体(TCR)的参与,通常会活化T细胞。专职APC也表达许多共刺激性分子,所述分子参与在T细胞上的其它受体,并促进活化。一种经证实的抑制T细胞活化的方式是,干扰这些共刺激性分子的参与。最值得注意的是,CTLA4-Ig可以用于阻止通过CD28分子的关键共刺激性信号,由此干扰T细胞应答。
已经鉴别出其它共刺激性分子,并且这些分子似乎在它们的反结构(counter-structure)被表达的特定情况下是重要的。一种这样的分子(仅在最近才理解其作用)是共刺激性受体CD226。CD226的反结构是连接素-家族蛋白PVR (CD155)和连接素-2 (CD112),它们在自身免疫疾病或自身炎性疾病中在通常被淋巴细胞浸润的组织中广泛地表达,包括上皮、内皮、滑膜细胞和中枢神经系统(CNS)的细胞。重要的是,已经证实,在非专职APC刺激T细胞的情况下,通过CD226的信号对于T细胞活化而言是关键性的(参见Gilfillan等人,J. Exp. Med. 205:2965-2973, 2008; Iguchi-Manaka等人,J. Exp. Med. 205:2959-2964,2008),所述情况可以包括自身免疫疾病,其中T细胞被活化,并在炎症组织中造成损伤。此外,最近已经将CD226的多态性与发展许多自身免疫病症的风险相关联,所述自身免疫病症包括多发性硬化(MS)、I型糖尿病、格雷夫斯氏病和韦格纳氏肉芽肿病(参见InternationalMultiple Sclerosis Genetics Consortium (IMSGC), Genes Immun. 10:11-14, 2009;Hafler等人,Genes Immun. 10:5-10, 2009; Maier和Hafler, Immunol. Rev. 229:322-336, 2009; Mait等人, “Non-synonymous variant (Gly307Ser) in CD226 isassociated with susceptibility to multiple autoimmune diseases,”Rheumatology (Oxford) (2010年3月24日[印刷之前的电子公开]))。因此,干扰免疫细胞功能中的CD226介导的信号,存在确实的合理性。
VSTM3 (在国际PCT公开号WO 06/124667中也称作B7R1)是CD28家族的一个抑制成员,其已经被证实也结合PVR和连接素-2,即与CD226相同的反结构。实际上,VSTM3和CD226交叉竞争与PVR和连接素-2的结合,这表明,它们结合重叠的(如果不是相同的)区域,尽管VSTM3似乎以稍微更高的亲和力结合。
已知许多细胞表面受体的可溶性形式。这些可溶性受体与它们的细胞表面对应物的配体结合结构域相对应。例如,天然存在的可溶性细胞因子受体抑制细胞因子应答,并起运输蛋白的作用。(参见,例如,Aggarwal和Puri, “Common and Uncommon Features ofCytokines and Cytokine Receptors:An Overview,”Aggarwal和Puri编, Human Cytokines: Their Role in Disease and Therapy, Blackwell Science, 1995, 3-24)。另外,已经发现,通过使用融合蛋白使可溶性受体多肽二聚化,可以增强这些可溶性受体的结合性质,使得它们变成它们的相应配体的治疗上有用的拮抗剂。这样的二聚融合体的代表是免疫球蛋白融合体。(参见,例如,Sledziewski等人, 美国专利号5,155,027和5,567,584; Jacobs等人, 美国专利号5,605,690; Wallner等人, 美国专利号5,914,111;和Ashkenazi和Chamow, Curr. Opin. Immunol. 9:195-200, 1997)。
例如,已经证实,可溶性的VSTM3在T-细胞介导的疾病的动物模型中是治疗上有效的。具体地,已经证实,可溶性的VSTM3-Fc二聚体(以常规的二价形式)在体内抑制T细胞应答(通过迟发型超敏(DTH)反应测得),并且这样的二聚体在胶原诱导的关节炎(CIA) 模型中减轻发病率和进展。(参见国际PCT公开号WO 06/124667)。还已经证实,一种可溶性的VSTM3-VASP四聚体在CIA模型中是有效的(参见相同出处)。
多种生物学效应(包括许多细胞类型的生长和分化)也由小的可溶性蛋白(统称为细胞因子)介导(参见,例如,Arai等人, Annu. Rev. Biochem. 59:783, 1990; Mosmann,Curr. Opin. Immunol. 3:311, 1991; Paul和Seder, Cell 76:241, 1994)。构成细胞因子集合的蛋白质包括:白介素、干扰素、集落刺激因子、肿瘤坏死因子和其它调节分子。
细胞表面受体和细胞因子的经证实的体内活性,说明了介导受体和细胞因子的生物活性的分子的临床潜力和对所述分子的需求。例如,促炎症性的受体和细胞因子的经证实的体内活性(包括共刺激性受体在介导免疫细胞应答中的活性),说明了促炎症性分子的拮抗剂的巨大临床潜力和对所述拮抗剂的需求。具体地,需要这样的细胞表面受体和细胞因子的可溶性融合体,其与已知的融合蛋白形式相比具有提高的活性。本文描述的发明满足了这些和其它需要。
发明内容
在一个方面,本发明提供了可溶性多肽融合体,其从氨基端至羧基端包括P1-L1-D-L2-P2或P2-L2-P1-L1-D,其中P1是(i) 第一种细胞表面受体的细胞外结构域或其功能变体或片段,或(ii) 第一种细胞因子或其功能变体或片段;L1是第一种多肽接头;D是二聚化结构域;L2是第二种多肽接头;且P2是(i) 第二种细胞表面受体的细胞外结构域或其功能变体或片段,或(ii) 第二种细胞因子或其功能变体或片段。在一个有关的方面,本发明提供了二聚蛋白,其包含第一种可溶性多肽融合体和第二种可溶性多肽融合体,其中所述第一种和第二种可溶性多肽融合体中的每一种包含上述的式P1-L1-D1-L2-P2或P2-L2-P1-L1-D。
在上述的可溶性多肽融合体或二聚蛋白的某些实施方案(其中P1是第一种细胞表面受体的细胞外结构域或其功能变体或片段,和/或P2是第二种细胞表面受体的细胞外结构域或其功能变体或片段)中,所述第一种细胞表面受体和/或所述第二种细胞表面受体各自选自:4-1BB;ACTH受体;激活素受体;BLTR (白三烯B4受体);BMP受体;C3a受体;C5a受体;CCR1;CCR2;CCR3;CCR4;CCR5;CCR6;CCR7;CCR8;CCR9;CD19;CD22;CD27;CD28;CD30;CD40;CD70;CD80;CD86;CTLA-4;CD226;VSTM3 (B7R1);CD112;CD155;B7H6;NKp30;ICAM;VLA-4;VCAM;CT-1受体;CX3CR1;CXCR1;CXCR2;CXCR3;CXCR4;CXCR5;D6;DARC;DcR3;DR4;DR5;DcR1;DcR2;ECRF3;Fas;fMLP受体;G-CSF受体;GIT受体;GM-CSF受体;生长激素受体;HVEM;BTLA;干扰素-α受体;干扰素-β受体;干扰素-γ受体;IL-1受体I型;IL-1受体II型;IL-10受体;IL-11受体;IL-12受体;IL-13受体;IL-15受体;IL-16受体(CD4);IL-17受体A;IL-17受体B;IL-17受体C;IL-17受体D;IL-17受体E;IL-18受体;IL-2受体;IL-3受体;IL-4受体;IL-5受体;IL-6受体;IL-7受体;IL-9受体;IL-20受体A;IL-20受体B;IL-21受体;IL-22受体A;IL-22受体B;IL-28受体A;IL-27受体A;IL-31-受体A;BCMA;TACI;BAFF受体;免疫调节性的脑信号蛋白受体CD72;卡波西肉瘤相关的疱疹病毒GPCR;脂氧素A4受体;淋巴毒素β受体;溶血磷脂生长因子受体;神经激肽1;内啡肽的 μ、δ和κ阿片受体;制癌蛋白M受体;骨桥蛋白受体;骨保护素;Ox40;PACAP和VIP受体;PAF受体;痘病毒;IFNα/β受体同系物;痘病毒IFNγ受体同系物;痘病毒IL-1β受体同系物;痘病毒膜结合的G蛋白偶联的受体同系物;痘病毒分泌的趋化因子结合蛋白;痘病毒TNF受体同系物;催乳素受体;RANK;RON受体;SCF受体;生长激素抑制素受体;T1/ST2;TGF-β受体;TNF受体(例如,p60和p80);TNFRSF19;TPO受体;US28;XCR1;促红细胞生成素受体;生长激素受体;白血病抑制因子受体;和C-kit受体。在P1和P2都是细胞表面受体的细胞外结构域或其功能变体或片段的情况下,所述第一种和第二种细胞表面受体可以是相同的或不同的。
在其它实施方案中,在P1是第一种细胞因子或其功能变体或片段和/或P2是第二种细胞因子或其功能变体或片段的情况下,所述第一种细胞因子和/或所述第二种细胞因子各自选自:α-MSH;9E3/cCAF;ACTH;激活素;AK155;血管生成抑制因子;Apo2L/TRAIL;APRIL;BAFF (BLys);BLR1配体/ BCA-1/BLC/CXCL13;BMP家族;BRAK;降钙素基因相关的肽(CGRP);传染性软疣病毒的CC趋化因子;CCL27;CCL28;CD100/Sema4D;CD27配体;CD30配体;CD40配体;CKβ8-1/MPIF-1/CCL23;CLF/CLC;CSF-1;CT-1;CTAP-III、βTG和NAP-2//CXCL7;CXCL16;防御素类;ELC/MIP-3β/Exodus-3/CCL19;ENA-78/CXCL5;内啡肽类;内皮抑素;嗜酸性粒细胞趋化因子2/MPIF-2/CCL24;嗜酸性粒细胞趋化因子/CCL11;促红细胞生成素;Exodus-1/LARC/MIP-3α(SCYA 20);Fas配体;Flt-3配体;fMLP;Fractalkine/CX3CL1;G-CSF;GCP-2/CXCL6;GM-CSF;生长激素;HCC-1/CCL14;HCC-4/CCL16;高速泳动族框1(HMGB1);人Cathelicidin抗微生物肽LL-37;I-309/CCL1;IFNα、IFNβ和IFNω配体;IFNγ;IL-1α;IL-1β;IL-10;IL-11;IL-12;IL-13;IL-15;IL-16;IL-17A;IL-17B;IL-17C;IL-17D;IL-17E;IL-17F;IL-18;IL-1Ra;IL-2;IL-27;IL-3;IL-4;IL-5;IL-6;IL-7;IL-8/CXCL8;IL-9;IP-10/CXCL10;IL-19;IL-20;IL-21;IL-22;IL-23;IL-24;IL-26;IL-31;角质形成细胞生长因子;KSHV-相关的IL-6配体;瘦素;白细胞诱素1/HCC-2/MIP-1δ/CCL15;白三烯B4;LIGHT;脂氧素;淋巴细胞趋化因子/XCL1;淋巴毒素α和β;溶血磷脂生长因子;巨噬细胞-衍生的趋化因子;巨噬细胞刺激蛋白(MSP);MCP-1/CCL2、MCP-2/CCL8、MCP-3/CCL7、MCP-4/CCL13和MCP-5/CCL12;甲氧雌二醇;MGSA/GRO/CXCL1、CXCL2、CXCL3;MIF;MIG/CXCL9;MIP-1α/CCL3、MIP-1β/CCL4;MIP-1γ/MRP-2/CCF18/CCL9/10;Mu C10/CCL6;制癌蛋白M;骨桥蛋白;副痘病毒(羊传染性口疮病毒) IL-10同系物;PARC/DC-CCK1/AMAC-1/CCL18;PDGF-A;PDGF-B;PDGF-C;PDGF-D;血小板活化因子;血小板因子4/CXCL4;与表皮生长因子有关的痘病毒生长因子;痘病毒分泌的补体调控蛋白;羊传染性口疮病毒的痘病毒血管内皮生长因子(VEGF)同系物;催乳素;RANK配体;RANTES/CCL5;S100A12;SDF-1/CXCL12;SERP-1、分泌型痘病毒丝氨酸蛋白酶抑制蛋白;SLC (6Ckine)/Exodus-2/TCA-4/CCL21;生长激素抑制素;干细胞因子;P物质;TARC/CCL17;TCA3/小鼠CCL1;TECK/CCL25;TGFβ;血小板生成素;TNFα;TSG-6;TWEAK;痘苗病毒脑信号蛋白;vCXC-1和vCXC-2;VEGF;VIP和PACAP;和病毒的IL-10变体。在P1和P2都是细胞因子或其功能变体或片段的情况下,所述第一种和第二种细胞因子可以是相同的或不同的。
在某些实施方案中,P1和P2中的每一种是VSTM3 (B7R1)的细胞外结构域或其功能变体或片段。例如,在具有上述的式P1-L1-D-L2-P2或P2-L2-P1-L1-D的可溶性多肽融合体的具体变化中,P1是与SEQ ID NO:2的氨基酸残基25-141具有至少95%同一性的第一种多肽,且P2是与SEQ ID NO:2的氨基酸残基25-141具有至少95%同一性的第二种多肽;其中所述多肽融合体能够特异性地结合CD155的细胞外结构域(SEQ ID NO:22的氨基酸残基28-343)。在有些实施方案中,P1和P2中的至少一种与SEQ ID NO:2的氨基酸残基25-141具有100%同一性。在其它变化中,P1和P2中的至少一种包括在与SEQ ID NO:2的残基69相对应的氨基酸位置处的非半胱氨酸残基,诸如酪氨酸。例如,在具体实施方案中,P1和P2中的至少一种具有在SEQ ID NO:18的残基23-139中所示的氨基酸序列。
根据上述多肽融合体或二聚蛋白使用的特别合适的二聚化结构域包括免疫球蛋白重链恒定区。例如,在具体的变化中,所述二聚化结构域D是Fc片段,诸如人γ1 Fc片段。
在上述的多肽融合体或二聚蛋白的有些实施方案中,所述接头L1由15-32个氨基酸残基组成,其中这些残基中的1-8个(例如,2个) 是半胱氨酸残基。在具体变化中,L1包含免疫球蛋白铰链区或其变体。例如,在一个具体实施方案中,L1包含免疫球蛋白铰链变体(例如,人γ1铰链变体),其中与Eu残基220相对应的半胱氨酸残基被丝氨酸替代。
根据上述多肽融合体或二聚蛋白使用的特别合适的L2接头包括这样的接头:所述接头包含多个甘氨酸残基,且任选地包含至少一个丝氨酸残基。例如,在包含式P1-L1-D-L2-P2的多肽融合体或包含第一种和第二种这样的多肽融合体的二聚蛋白的一个具体实施方案中,L2包含在SEQ ID NO:25中所示的氨基酸序列。在包含式P2-L2-P1-L1-D的多肽融合体或包含第一种和第二种这样的多肽融合体的二聚蛋白的一个具体实施方案中,L2包含式[Gly-Gly-Gly-Ser]n (SEQ ID NO:26),其中n是3-5的整数,包括端点。
在包含式P1-L1-D-L2-P2或P2-L2-P1-L1-D(且其中P1和P2中的每一种是VSTM3的细胞外结构域或其功能变体或片段)的多肽融合体的具体变化中,所述多肽融合体包含选自下述的氨基酸序列:(a) 在SEQ ID NO:18的残基23-493中所示的氨基酸序列,和(b) 在SEQ ID NO:20的残基23-508或23-507中所示的氨基酸序列。类似地,在根据本发明的二聚蛋白的具体变化中,所述第一种和第二种多肽融合体中的每一种包含选自下述的氨基酸序列:(a) 在SEQ ID NO:18的残基23-493中所示的氨基酸序列,和(b) 在SEQ ID NO:20的残基23-508或23-507中所示的氨基酸序列。
在另一个方面,本发明提供了多核苷酸,其编码上述的多肽融合体。在一个有关的方面,本发明提供了载体,其包含这样的多核苷酸。例如,在有些实施方案中,本发明提供了表达载体,其包含下述可操作地连接的元件:转录启动子;编码上述多肽融合体的DNA区段;和转录终止子。
在其它有关的方面,本发明提供了培养的包含这种载体的细胞,以及用于生产如上公开的多肽或二聚蛋白的方法。例如,在有些实施方案中,根据本发明的培养的细胞包含表达载体,所述表达载体包含下述可操作地连接的元件:转录启动子;编码上述多肽融合体的DNA区段;和转录终止子;且其中所述细胞表达由所述DNA区段编码的多肽融合体。在制备所述多肽融合体的方法的某些变化中,所述方法包括:(i) 培养包含如上公开的表达载体的细胞,其中所述细胞表达由所述DNA区段编码的多肽融合体,并生产编码的多肽融合体;和(ii) 回收所述可溶性多肽融合体。类似地,在制备二聚蛋白的方法的某些变化中,所述方法包括:(i) 培养包含如上公开的表达载体的细胞,其中所述细胞表达由所述DNA区段编码的多肽融合体,并生产编码的多肽融合体作为二聚蛋白;和(ii) 回收所述二聚蛋白。
本发明另外包括组合物,所述组合物包含上述的多肽融合体或二聚蛋白和至少一种药学上可接受的载体。
在另一个方面,本发明提供了治疗T-细胞介导的免疫障碍的方法,其中使用如上公开的二聚的VSTM3蛋白。所述方法通常包括,给具有T-细胞介导的免疫障碍的受试者施用有效量的二聚的VSTM3蛋白。适合用本文公开的二聚的VSTM3蛋白治疗的T-细胞介导的免疫障碍包括,例如,自身免疫疾病、移植物抗宿主病(GVHD)和移植排斥。在具体变化中,所述方法是治疗选自下述的自身免疫病的方法:类风湿性关节炎、多发性硬化(MS) (例如,脊髓-视觉多发性硬化、原发进行性多发性硬化(PPMS)和复发缓解型多发性硬化(RRMS))、胰岛素依赖性的糖尿病(IDDM)、系统性红斑狼疮(SLE)、腹部疾病、神经炎、多肌炎、银屑病、银屑病关节炎、白癫风、干燥综合征、自身免疫胰腺炎、炎性肠病(例如,克罗恩氏病、溃疡性结肠炎)、活动性慢性肝炎、肾小球肾炎、硬皮病、结节病、自身免疫性甲状腺病、桥本甲状腺炎、格雷夫斯病、韦格纳氏肉芽肿病、重症肌无力、哮喘、阿狄森氏病、自身免疫性葡萄膜炎、寻常型天疱疮、原发性胆汁性肝硬化、恶性贫血、交感性眼炎(sympathetic opthalmia)、葡萄膜炎、自身免疫性溶血性贫血、肺纤维化、慢性铍病和特发性肺纤维化。
参考下述发明详述和附图,将会明白本发明的这些和其它方面。
定义
除非另外定义,在本文中使用的所有技术和科学术语具有与所述方法和组合物有关的领域的普通技术人员通常理解相同的含义。本文使用的下述术语和短语具有指定给它们的含义,除非另有说明。
术语“一个(a)”、“一种(an)”和“所述(the)”包括复数指示物,除非上下文另外清楚地指出。
“多肽”是由肽键连接的氨基酸残基的多聚体,无论是天然产生的还是通过合成产生的。少于约10个氨基酸残基的多肽通常称为“肽”。
“蛋白质”是包含一个或多个多肽链的大分子。蛋白质也可包含非肽组分,例如糖基。产生蛋白质的细胞可以给该蛋白质添加糖和其它非肽取代基,并且所述糖和其它非肽取代基将随细胞类型而变化。在本文中通过它们的氨基酸骨架结构定义蛋白质;一般不指明取代基(例如糖基),虽然如此,但其可存在。
术语“氨基端”(或“N-端”)和“羧基端”(或“C-端”) 在本文中用于表示在多肽内的位置。在上下文允许的情况下,关于参照多肽的特定序列或部分,使用这些术语来表示临近或相对位置。例如,在多肽内位于参照序列的羧基端的特定序列位于参照序列的羧基末端近端,但不一定在完整多肽的羧基末端处。
当应用于序列中的氨基酸残基的位置时,术语“与……相对应”是指,当最佳地比对多个序列时,在所述序列中的对应位置。
多肽或蛋白之间的“非共价结合”包括氢键键合、空间相互作用、疏水相互作用和离子相互作用。
术语“多核苷酸”和“核酸”在本文中同义地使用,且表示从5'至3'端读出的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双链聚合物。多核苷酸包括RNA和DNA,且可以从天然来源分离,在体外合成,或从天然的和合成的分子的组合制备。多核苷酸的大小表示为碱基对(缩写“bp”)、核苷酸(“nt”)或千碱基(“kb”)。在上下文允许的情况下,后2个术语可以描述单链的或双链的多核苷酸。当所述术语应用于双链分子时,它用于表示总长度,且将理解为与术语“碱基对”等价。本领域技术人员会认识到,双链多核苷酸的2条链的长度可以稍有差异,且作为酶切割的结果,它们的末端可以是交错的;因而,在双链多核苷酸分子内的所有核苷酸可以未配对。这样的未配对的末端的长度通常不超过20 nt。
“区段”是更大分子(例如,多核苷酸或多肽)的具有指定属性的部分。例如,编码指定多肽的DNA区段是更长DNA分子(诸如质粒或质粒片段)的一部分,当从5’至3’方向读出时,该部分编码指定多肽的氨基酸序列。并且,在根据本发明的多肽融合体的背景下,与细胞因子或细胞表面受体的细胞外结构域相对应的多肽区段是更长多肽融合体分子的一部分,除了所述与细胞因子或细胞表面受体的细胞外结构域相对应的多肽区段以外,所述多肽融合体分子还包括本文所述的其它多肽区段(例如,接头、二聚化结构域)。
本文使用的“单体”或“单体结构域”是指,与细胞因子或细胞表面受体的细胞外结构域相对应的多肽区段。
术语“表达载体”用于表示线性的或环状的DNA分子,所述DNA分子包含编码目标多肽的区段,所述区段与提供它的转录的额外区段可操作地连接。这样的额外区段包括启动子和终止子序列,且也可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体通常源自质粒或病毒DNA,或可以含有二者的元件。
术语“启动子”在本文中以它的本领域公知的含义进行使用,以表示含有DNA序列的基因部分,所述DNA序列提供RNA聚合酶的结合和转录起始。启动子序列通常(但并非总是)存在于基因的5' 非编码区。
术语“分泌信号序列”是编码多肽(“分泌肽”)的DNA序列,所述多肽作为更大多肽的组分,引导该更大多肽穿过合成它的细胞的分泌途径。在运输穿过分泌途径的过程中,通常切割更大的肽以除去分泌肽。
“可操作地连接”是指,2个或更多个实体连接到一起,使得它们为了它们的预期目的协同地起作用。当表示DNA区段时,所述短语指示,例如,编码序列连接在正确的读码框中,且转录在启动子中开始,并前进穿过所述区段到达终止子。当表示多肽时,“可操作地连接”包括共价地(例如,通过二硫键键合)和非共价地(例如,通过氢键键合、疏水相互作用或盐桥相互作用)连接的序列,其中保留所述序列的所需功能。
“分离的多肽”是这样的多肽:其基本上不含在天然状态下与该多肽结合的污染性细胞组分,例如糖、脂质或其它蛋白性的杂质。通常,分离的多肽的制剂含有高度纯化形式(即,至少约80%纯度、至少约90%纯度、至少约95%纯度、大于95%纯度,诸如96%、97%或98%或或更高的纯度或高于99%的纯度)的该多肽。显示含有分离的多肽的特定蛋白制剂一种方法是,对蛋白制剂进行十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶电泳和凝胶的考马斯亮蓝染色以后,显现出单一条带。然而,术语“分离的”不排除相同的多肽以替代性的物理形式(诸如二聚体,或可替换地糖基化或衍生化形式)存在。
“免疫球蛋白”是在脊椎动物生物体中起抗体作用的血清蛋白。已经在高等脊椎动物中鉴别出了5类“免疫球蛋白”或抗体、蛋白(IgG、IgA、IgM、IgD和IgE)。IgG构成主要类别;它通常作为在血浆中发现的第二最丰富的蛋白存在。在人类中,IgG由4个亚类组成,所述亚类命名为IgG1、IgG2、IgG3和IgG4。用希腊符号γ表示IgG类的重链恒定区。例如,IgG1亚类的免疫球蛋白含有γ1重链恒定区。每个免疫球蛋白重链具有由恒定区蛋白结构域(CH1、铰链、CH2和CH3) 组成的恒定区,所述恒定区蛋白结构域对于物种的给定亚类而言基本上不变。编码人和非人免疫球蛋白链的DNA序列是本领域已知的。参见,例如,Ellison等人, DNA1:11-18, 1981; Ellison等人, Nuc. Acids Res. 10:4071-4079, 1982; Kenten等人,Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Seno等人, Nuc. Acids Res. 11:719-726, 1983; Riechmann等人, Nature 332:323-327, 1988; Amster等人, Nuc.Acids Res. 8:2055-2065, 1980; Rusconi和Kohler, Nature 314:330-334, 1985; Boss等人, Nuc. Acids Res. 12:3791-3806, 1984; Bothwell等人, Nature 298:380-382,1982; van der Loo等人, Immunogenetics 42:333-341, 1995; Karlin等人, J. Mol.Evol. 22:195-208, 1985; Kindsvogel等人, DNA 1:335-343, 1982; Breiner等人,Gene 18:165-174, 1982; Kondo等人, Eur. J. Immunol. 23:245-249, 1993;和GenBank登记号J00228。关于免疫球蛋白结构和功能的综述,参见Putnam, The Plasma Proteins,Vol V, Academic Press, Inc., 49-140, 1987;和Padlan, Mol. Immunol. 31:169-217,1994。
“免疫球蛋白铰链”是免疫球蛋白重链的连接可变结构域和CH1结构域的部分。在SEQ ID NO:27内,所述铰链是约残基99-113 (在图1A中所示的Eu残基216-230)。
本文使用的术语“Fc片段”、“Fc区”、或“Fc结构域”是同义的,且表示抗体的负责与细胞上的抗体受体和补体的C1q组分结合的部分。Fc代表“结晶性的片段”,即抗体的可容易地形成蛋白晶体的片段。独特蛋白片段(它们最初通过蛋白水解消化来描述)可以定义免疫球蛋白蛋白质的总体一般结构。如在文献中最初定义的,Fc片段由二硫键连接的重链铰链区、CH2和CH3结构域组成。但是,新近已经将该术语应用于由CH3、CH2和铰链的至少一部分组成的单链,其足以与第二个这样的链形成二硫键连接的二聚体。本文使用的术语Fc包括天然地存在的序列的变体。
本文使用的“二聚化结构域”表示这样的多肽:其具有对第二种多肽的亲和力,使得所述两种多肽在生理条件下结合以形成二聚体。所述第二种多肽可以是相同的或不同的多肽。所述多肽可以通过共价和/或非共价结合而彼此相互作用。二聚化结构域的实例包括:Fc区;铰链区;CH3结构域;CH4结构域;CH1或CL结构域;亮氨酸拉链结构域(例如,jun/fos亮氨酸拉链结构域,参见,例如,Kostelney等人, J. Immunol., 148:1547-1553,1992;或酵母GCN4亮氨酸拉链结构域);异亮氨酸拉链结构域;二聚化细胞表面受体(例如,白介素-8受体(IL-8R);或亲和素异源二聚体诸如LFA-1或GPIIIb/IIIa) 的二聚化区域;分泌型二聚化配体(例如,神经生长因子(NGF)、神经营养因子-3 (NT-3)、白介素-8 (IL-8)、血管内皮生长因子(VEGF)或脑-衍生的神经营养因子(BDNF);参见,例如,Arakawa等人, J.Biol. Chem. 269:27833-27839, 1994,和Radziejewski等人, Biochem. 32:1350, 1993)的二聚化区域;和这样的多肽,其包含至少一个半胱氨酸残基(例如,约1、2或3至约10个半胱氨酸残基),使得二硫键可以形成在所述多肽与包含至少一个半胱氨酸残基的第二种多肽之间(在下文中称作“合成铰链”)。根据本发明的一种优选的二聚化结构域是Fc区。
术语“接头”或“多肽接头”在本文中用于表示通过肽键连接且连接2个不连续的、单独的多肽区域的2个或更多个氨基酸。所述接头通常设计成允许所述单独的多肽区域行使它们各自的功能(例如,在与其它多肽区域相连的二聚化结构域的情况下,与另一个对应的二聚化结构域结合以形成二聚体)。所述接头可以是天然序列、其变体或合成的序列的一部分。在本文中,也使用缩写“L”表示接头。与“L”在一起的下标(例如,“1”或“2”)在本文中用于区分在多肽链内的多个接头,就氨基酸序列而言,所述接头可以是相同的或不同的。
术语“变体VSTM3基因”或“变体B7R1基因”表示编码具有特定氨基酸序列的多肽的核酸分子,所述氨基酸序列是SEQ ID NO:2的变体。这些变体包括VSTM3 (B7R1) 基因的天然存在的多态性,以及含有SEQ ID NO:2的氨基酸序列的保守氨基酸置换的合成基因。VSTM3基因的其它变体形式是含有本文所述核苷酸序列的插入或缺失的核酸分子。可以如下鉴定变体VSTM3基因:例如,在严谨条件下,确定该基因是否与具有SEQ ID NO:1的核苷酸序列的核酸分子或它的互补序列杂交。
可替换地,可通过序列比较来鉴定变体VSTM3基因。当就最大对应性进行比对时,如果两个氨基酸序列的氨基酸残基相同,那么这两个氨基酸序列具有“100%氨基酸序列同一性”。类似地,当就最大对应性进行比对时,如果两个核苷酸序列的核苷酸残基相同,那么这两个核苷酸序列具有“100%核苷酸序列同一性”。使用标准的软件程序,例如由DNASTAR(Madison, 威斯康辛州)生产的LASERGENE生物信息学计算套件中包含的程序,可以进行序列比较。用于通过确定最优比对来比较两个核苷酸或氨基酸序列的其它方法,是本领域技术人员众所周知的。(参见,例如,Peruski和Peruski, The Internet and the NewBiology: Tools for Genomic and Molecular Research (ASM Press, Inc. 1997);Wu等人, (编), “Information Superhighway and Computer Databases of Nucleic Acidsand Proteins,”Methods in Gene Biotechnology 123-151 (CRC Press, Inc. 1997);Bishop (编), Guide to Human Genome Computing (第2版, Academic Press, Inc.1998))。如果2个序列相对于彼此具有至少80%、至少90%或至少95%序列同一性,则认为这2个核苷酸或氨基酸序列具有“实质上类似的序列同一性”或“实质的序列同一性”。下文描述用于确定序列同一性的特定方法。
不管用于鉴定变体VSTM3基因或变体VSTM3多肽的特定方法如何,可功能性地表征变体基因或由变体基因编码的多肽的特异性地结合抗-VSTM3抗体的能力。使用本文描述的生物学或生物化学测定法,通过多肽的结合CD155的能力,也可功能性地表征变体VSTM3基因或变体VSTM3多肽。
术语“等位基因变体”在本文中用于表示占据相同染色体基因座的基因的两种或更多种替代形式中的任何一种。等位基因变异通过突变天然地产生,并可导致群体内的表型多态性。基因突变可以是沉默的(在编码的多肽中无变化),或可以编码具有改变的氨基酸序列的多肽。术语等位基因变体在本文中也用于表示由基因的等位基因变体编码的蛋白质。
术语“直系同源物”表示从一个物种获得的这样的多肽或蛋白质:所述多肽或蛋白质是来自不同物种的多肽或蛋白质的功能性对应物。直系同源物之间的序列差异是物种形成的结果。
本文使用的术语“T-细胞介导的免疫障碍”表示,具有至少部分地由T细胞活性介导的病理学的任意疾病或障碍。T-细胞介导的免疫障碍包括,例如,自身免疫疾病(例如,类风湿性关节炎、多发性硬化)、移植物抗宿主病(GVHD)和移植排斥。除了主要通过T-细胞免疫应答来表征的疾病以外,T-细胞介导的免疫障碍另外包括,例如,T-细胞依赖性的B-细胞介导的适应症,例如,抗体介导的自身免疫。这样的疾病或障碍特别适合使用本发明的二聚的VSTM3 (B7R1) 蛋白的治疗方法,如本文进一步所述。
在通过给本文所述的受试者施用可溶性二聚蛋白来治疗疾病的背景下,术语“有效量”表示,足以抑制所述疾病的一种或多种症状的出现或改善所述症状的这种分子的量。例如,在通过给本文所述的受试者施用可溶性的二聚VSTM3 (B7R1) 蛋白来治疗T-细胞介导的免疫障碍的具体背景下,术语“有效量”表示这样的这种分子的量:所述量足以调控受试者中的T-细胞介导的应答,从而抑制T-细胞介导的免疫障碍的一种或多种症状的出现或改善所述症状。根据本发明的方法,在“有效方案”中施用有效量的药剂。术语“有效方案”表示,施用的药剂的量和给药频率的组合,所述组合足以完成疾病的治疗或预防。
由于标准分析方法的不精确性,聚合物的分子量和长度理解为近似值。当这样的值表达为“约”X或“近似”X时,所述的X的值应理解为精确至±10%。
附图简要说明
图1A-1B解释了代表性的人免疫球蛋白γ1重链的一部分的氨基酸序列(SEQ IDNO:27) (基于Ellison等人,Nucl. Acids Res. 10:4071, 1982)。氨基酸序列编号是基于Eu索引(Edelman等人,Proc. Natl. Acad. Sci. USA 63:78-85, 1969; Kabat等人, Sequences of Proteins of Immunological Interest, US Department of Health andHuman Services, NIH, Bethesda, MD, 1991)。指示了通常参与轻链恒定区(LC)和重链恒定区(HC)的二硫键键合的Cys残基。显示了CH1、铰链、CH2和CH3结构域的边界。
图2A-2C解释了某些免疫球蛋白Fc多肽的氨基酸序列。氨基酸序列编号是基于Eu索引(Kabat等人,Sequences of Proteins of Immunological Interest, US Departmentof Health and Human Services, NIH, Bethesda, 1991)。解释的序列包括野生型人序列(“wt”;SEQ ID NO:28)和5个变体序列,所述变体序列命名为Fc-488 (SEQ ID NO:29)、Fc4(SEQ ID NO:30)、Fc5 (SEQ ID NO:31)、Fc6 (SEQ ID NO:32)和Fc7 (SEQ ID NO:33)。指示了通常参与轻链恒定区(LC)和重链恒定区(HC)的二硫键键合的Cys残基。“.”指示在该位置与野生型的同一性。***指示终止密码子;C-端Lys残基已经从Fc6除去。显示了铰链、CH2和CH3结构域的边界。
图3A-3F描绘了通过降低的体外增殖活性测得的、由可溶性B7R1 (VSTM3) 蛋白诱导的T细胞抑制。如在实施例13中所述,在有抗-CD3抗体和表达PVR的P815细胞存在下,在有或没有可溶性B7R1 (VSTM3)存在下,孵育得自3个供体的人T细胞。试验3种不同的可溶性B7R1蛋白的抑制CD4+ (图3A-3C)或CD8+ (图3D-3F) T细胞的增殖的能力。试验了3种不同的可溶性B7R1蛋白:B7r1(G25-P141) C69Y Fc5 barbell、B7r1(G25-P141) C69Y Fc5tandem和B7r1-Fc5。
图4描绘了mB7R1-Barbell治疗对实验性变应性脑脊髓炎(EAE)疾病评分的降低。如在下文中的实施例14所述,在小鼠中建立EAE。用单独的媒介(PBS),或用含有可溶性B7R1barbell构建体(mB7R1-Barbell)、二聚的鼠Fc2构建体(mB7R1-Fc2)或VASP构建体(mB7R1-VASP)的媒介,治疗小鼠(参见实施例14)。每个点代表每组n = 10-12只小鼠的平均值±SEM。B7R1-Barbell治疗组的疾病评分不同于PBS治疗的对照组的疾病评分,** 通过重复测量的方差分析(repeated measures ANOVA),p < 0.05。
图5描绘了mB7R1-Barbell和mB7R1-Tandem治疗对关节炎疾病评分的降低。如在下文中的实施例17所述,在小鼠中建立胶原诱导的关节炎(CIA)。用单独的媒介(PBS),或用含有可溶性B7R1 barbell构建体(mB7R1-Barbell)、tandem构建体(mB7R1-Tandem)或VASP构建体(mB7R1-VASP)的媒介,治疗小鼠(参见实施例17)。每个点代表每组n = 15只小鼠的平均值±SEM。B7R1-Barbell和B7R1-Tandem治疗组的疾病评分(爪厚度)不同于PBS治疗的对照组的疾病评分,* 通过双因素方差分析(two-way ANOVA),p < 0.05。
发明详述
I. 概述
本发明提供了与可溶性的二聚融合蛋白有关的组合物和方法。通常,本发明的二聚融合蛋白包含经由二聚化结构域连接的第一种和第二种多肽链,每个多肽链包含与细胞因子或细胞表面受体的细胞外结构域(在本文中也称作“单体”或“单体结构域”)相对应的第一种和第二种多肽区段。因此,所述二聚融合蛋白通常就单体结构域而言是四价的。每个多肽链的第一种和第二种单体结构域可以是相同的或不同的。在某些实施方案中,所述二聚融合蛋白的单体结构域是相同的,从而提供就特定单体结构域而言四价的分子。所述第一种和第二种单体结构域可以分别位于二聚化结构域的氨基端和羧基端(在本文中也称作“杠铃(barbell)”形式)。可替换地,所述第一种和第二种单体结构域可以串联地都位于二聚化结构域的羧基端或氨基端(在本文中也称作“串联(tandem)”形式)。
在特定方面,本发明提供了与可溶性的二聚VSTM3 (B7R1) 融合蛋白有关的组合物和方法,以及它们用于治疗T细胞介导的免疫障碍、特别是T-细胞介导的自身免疫疾病的用途。根据本发明,所述二聚的VSTM3融合体包含经由二聚化结构域连接的第一种和第二种多肽链,每个多肽链包含与VSTM3的细胞外结构域相对应的第一种和第二种单体结构域。因此,所述二聚的VSTM3融合体通常就VSTM3反受体(CD155)的结合位点而言是四价的。与VSTM3细胞外结构域相对应的第一种和第二种单体结构域可以以如上总结的杠铃或串联形式定位。如在本文中进一步所述,根据本发明的二聚的VSTM3融合蛋白在体内可特别有效地抑制T-细胞介导的应答。
II. 多肽融合体和二聚蛋白
因此,在一个方面,本发明提供了可溶性多肽融合体,其从氨基端至羧基端包括选自P1-L1-D-L2-P2和P2-L2-P1-L1-D的式,其中P1是(i) 第一种细胞表面受体的细胞外结构域或其功能变体或片段,或(ii) 第一种细胞因子或其功能变体或片段;L1是第一种多肽接头;D是二聚化结构域;L2是第二种多肽接头;且P2是(i) 第二种细胞表面受体的细胞外结构域或其功能变体或片段,或(ii) 第二种细胞因子或其功能变体或片段。
在本发明的某些实施方案中,P1和P2是相同的(或源自相同的细胞表面受体或细胞因子),从而在多肽融合体的同源二聚化以后,提供就P1/P2单体而言四聚的蛋白。
在有些替代实施方案中,P1和P2源自不同的细胞表面受体和/或细胞因子,从而在多肽融合体的同源二聚化以后,提供就2种不同的单体结构域(P1和P2)而言二聚的蛋白。例如,在有些变化中,P1与第一种细胞表面受体的细胞外结构域相对应,且P2与第二种细胞表面受体的细胞外结构域(其不同于所述第一种)相对应,使得所述融合体的同源二聚化产生就2种不同的细胞表面受体或其变体或片段而言同源二聚的蛋白。类似地,在其它变化中,P1与第一种细胞因子相对应,且P2与第二种细胞因子(其不同于所述第一种)相对应,使得所述融合体的同源二聚化产生就2种不同的细胞因子或其变体或片段而言同源二聚的蛋白。在其它变化中,P1与细胞表面受体的细胞外结构域相对应,且P2与细胞因子相对应,或反之亦然,使得所述融合体的同源二聚化产生就细胞表面受体和细胞因子或它们的变体或片段而言同源二聚的蛋白。
在其它实施方案中,P1和P2与得自异源二聚的细胞表面受体的2种不同亚基的细胞外结构域相对应,或者可替换地,与异源二聚的细胞因子的2种不同亚基相对应。在这样的实施方案中,将多肽接头L1和L2设计成,在单个多肽融合体内的单体结构域P1和P2之间提供足够的空间和柔性,以允许这些单体结构域彼此非共价地结合,从而形成单个功能性的异源二聚单元,使得所述融合体的同源二聚化产生具有2个这样的功能性的异源二聚单元的蛋白。
可以衍生出P1和/或P2的细胞表面受体的实例包括,例如,4-1BB;ACTH受体;激活素受体;BLTR (白三烯B4受体);BMP受体;C3a受体;C5a受体;CCR1;CCR2;CCR3;CCR4;CCR5;CCR6;CCR7;CCR8;CCR9;CD19;CD22;CD27;CD28;CD30;CD40;CD70;CD80;CD86;CTLA-4;CD226;VSTM3 (B7R1);CD112;CD155;B7H6;NKp30;ICAM;VLA-4;VCAM;CT-1受体;CX3CR1;CXCR1;CXCR2;CXCR3;CXCR4;CXCR5;D6;DARC;DcR3;DR4;DR5;DcR1;DcR2;ECRF3;Fas;fMLP受体;G-CSF受体;GIT受体;GM-CSF受体;生长激素受体;HVEM;BTLA;干扰素-α受体;干扰素-β受体;干扰素-γ受体;IL-1受体I型;IL-1受体II型;IL-10受体;IL-11受体;IL-12受体;IL-13受体;IL-15受体;IL-16受体(CD4);IL-17受体A (IL-17RA);IL-17受体B (IL-17RB);IL-17受体C (IL-17RC);IL-17受体D (IL-17RD);IL-17受体E (IL-17RE);IL-18受体;IL-2受体;IL-3受体;IL-4受体;IL-5受体;IL-6受体;IL-7受体;IL-9受体;IL-20受体A (IL-20RA);IL-20受体B (IL-20RB);IL-21受体;IL-22受体A (IL-22RA);IL-22受体B (IL-22RB);IL-28受体A (IL-28RA);IL-27受体A (IL-27RA);IL-31-受体A (IL-28RA);BCMA;TACI;BAFF受体;免疫调节性的脑信号蛋白受体CD72;卡波西肉瘤相关的疱疹病毒GPCR;脂氧素A4受体;淋巴毒素β受体;溶血磷脂生长因子受体;神经激肽1;内啡肽的 μ、δ和κ阿片受体;制癌蛋白M受体;骨桥蛋白受体;骨保护素;Ox40;PACAP和VIP受体;PAF受体;痘病毒;IFNα/β受体同系物;痘病毒IFNγ受体同系物;痘病毒IL-1β受体同系物;痘病毒膜结合的G蛋白偶联的受体同系物;痘病毒分泌的趋化因子结合蛋白;痘病毒TNF受体同系物;催乳素受体;RANK;RON受体;SCF受体;生长激素抑制素受体;T1/ST2;TGF-β受体;TNF受体(例如,p60和p80);TNFRSF19;TPO受体;US28;XCR1;促红细胞生成素受体;生长激素受体;白血病抑制因子受体;和C-kit受体。
可以衍生出P1和/或P2的细胞因子的实例包括,例如,α-MSH;9E3/cCAF;ACTH;激活素;AK155;血管生成抑制因子;Apo2L/TRAIL;APRIL;BAFF (BLys);BLR1配体/ BCA-1/BLC/CXCL13;BMP家族;BRAK;降钙素基因相关的肽(CGRP);传染性软疣病毒的CC趋化因子;CCL27;CCL28;CD100/Sema4D;CD27配体;CD30配体;CD40配体;CKβ8-1/MPIF-1/CCL23;CLF/CLC;CSF-1;CT-1;CTAP-III、βTG和NAP-2//CXCL7;CXCL16;防御素类;ELC/MIP-3β/Exodus-3/CCL19;ENA-78/CXCL5;内啡肽类;内皮抑素;嗜酸性粒细胞趋化因子2/MPIF-2/CCL24;嗜酸性粒细胞趋化因子/CCL11;促红细胞生成素;Exodus-1/LARC/MIP-3α(SCYA 20);Fas配体;Flt-3配体;fMLP;Fractalkine/CX3CL1;G-CSF;GCP-2/CXCL6;GM-CSF;生长激素;HCC-1/CCL14;HCC-4/CCL16;高速泳动族框1 (HMGB1);人Cathelicidin抗微生物肽LL-37;I-309/CCL1;IFNα、IFNβ和IFNω配体;IL-1α;IL-1β;IL-10;IL-11;IL-12;IL-13;IL-15;IL-16;IL-17A;IL-17B;IL-17C;IL-17D;IL-17E;IL-17F;IL-18;IL-1Ra;IL-2;IL-27;IL-3;IL-4;IL-5;IL-6;IL-7;IL-8/CXCL8;IL-9;IP-10/CXCL10;IL-19;IL-20;IL-21;IL-22;IL-23;IL-24;IL-26;IL-31;角质形成细胞生长因子;KSHV-相关的IL-6配体;瘦素;白细胞诱素1/HCC-2/MIP-1δ/CCL15;白三烯B4;LIGHT;脂氧素;淋巴细胞趋化因子/XCL1;淋巴毒素α和β;溶血磷脂生长因子;巨噬细胞-衍生的趋化因子;巨噬细胞刺激蛋白(MSP);MCP-1/CCL2、MCP-2/CCL8、MCP-3/CCL7、MCP-4/CCL13和MCP-5/CCL12;甲氧雌二醇;MGSA/GRO/CXCL1、CXCL2、CXCL3;MIF;MIG/CXCL9;MIP-1α/CCL3、MIP-1β/CCL4;MIP-1γ/MRP-2/CCF18/CCL9/10;MuC10/CCL6;制癌蛋白M;骨桥蛋白;副痘病毒(羊传染性口疮病毒) IL-10同系物;PARC/DC-CCK1/AMAC-1/CCL18;PDGF-A;PDGF-B;PDGF-C;PDGF-D;血小板活化因子;血小板因子4/CXCL4;与表皮生长因子有关的痘病毒生长因子;痘病毒分泌的补体调控蛋白;羊传染性口疮病毒的痘病毒血管内皮生长因子(VEGF)同系物;催乳素;RANK配体;RANTES/CCL5;S100A12;SDF-1/CXCL12;SERP-1、分泌型痘病毒丝氨酸蛋白酶抑制蛋白;SLC (6Ckine)/Exodus-2/TCA-4/CCL21;生长激素抑制素;干细胞因子;P物质;TARC/CCL17;TCA3/小鼠CCL1;TECK/CCL25;TGFβ;血小板生成素;TNFα;TSG-6;TWEAK;痘苗病毒脑信号蛋白;vCXC-1和vCXC-2;VEGF;VIP和PACAP;和病毒的IL-10变体。
可以如下容易地鉴定细胞表面受体的特定细胞外结构域的功能变体或片段:使用常规的生物学或生化测定法,所述测定法用于评估所述变体或片段的特异性地结合所述细胞表面受体的相应配体或反受体的能力。类似地,可以如下容易地鉴定特定细胞因子的功能变体或片段:使用常规的生物学或生化测定法,所述测定法用于评估所述变体或片段的特异性地结合所述细胞因子的相应受体的能力。
特定参照多肽(例如,野生型细胞因子或野生型细胞表面受体的细胞外结构域,例如,VSTM3(B7R1)的细胞外结构域)的功能变体通常被表征为,与参照多肽相比具有一个或多个氨基酸置换、缺失或添加。这些变化优选地具有次要性质(minor nature),也就是说,保守氨基酸置换(参见,例如,下文中的表1,该表列出了一些示例性的保守氨基酸置换)和不会显著影响蛋白或多肽的折叠或活性的其它置换;小缺失,通常为1至约30个氨基酸;和小氨基端或羧基端延伸,诸如氨基端甲硫氨酸残基,最多约20-25个残基的小接头肽,或便利纯化的小延伸(亲和标签),诸如聚-组氨酸段、蛋白A (Nilsson等人, EMBO J. 4:1075,1985; Nilsson等人, Methods Enzymol. 198:3, 1991)、谷胱甘肽S转移酶(Smith和Johnson, Gene 67:31, 1988),或其它抗原表位或结合结构域。(一般参见Ford等人, Protein Expression and Purification 2:95-107, 1991)。编码亲和标签的DNA可从商业供应商(例如,Pharmacia Biotech, Piscataway, NJ)得到。
表1:保守氨基酸置换
根据本领域已知的方法,例如定点诱变或丙氨酸扫描诱变(Cunningham和Wells,Science 244:1081-1085, 1989; Bass等人, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991),可以鉴定受体或细胞因子多肽中的必需氨基酸。在后一种技术中,在分子中的每一个残基处引入单个丙氨酸突变,并且试验所得的突变分子的生物学活性(例如,配体结合和信号转导),以鉴定对于该分子的活性至关重要的氨基酸残基。通过分析晶体结构,还可以确定配体-受体相互作用的位点,所述晶体结构通过诸如核磁共振、晶体学或光亲和标记等技术来确定。(参见,例如,de Vos等人, Science 255:306-312, 1992; Smith等人, J. Mol. Biol. 224:899-904, 1992; Wlodaver等人, FEBS Lett. 309:59-64, 1992)。通过分析与有关的受体或细胞因子的同源性,也可以推断出必需氨基酸的身份。
使用已知的诱变和筛选方法,例如由Reidhaar-Olson和Sauer (Science 241:53-57, 1988)或Bowie和Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989)公开的方法,可以产生并试验多个氨基酸置换。简而言之,这些作者公开了这样的方法:所述方法用于同时随机化多肽中的两个或更多个位点,选择功能性多肽,然后对诱变处理的多肽测序,以确定各位点处可允许的置换的谱。可使用的其它方法包括:噬菌体展示法(例如,Lowman等人, Biochem. 30:10832-10837, 1991; Ladner等人, 美国专利号5,223,409;Huse, WIPO公开WO 92/06204),和区域定点诱变(Derbyshire等人, Gene 46:145, 1986;Ner等人, DNA 7:127, 1988)。
通过DNA改组(参见,例如,Stemmer, Nature 370:389, 1994; Stemmer, Proc. Nat’l Acad. Sci. USA 91:10747, 1994;国际公开号WO 97/20078),也可产生变体核苷酸和多肽序列。简而言之,如下产生变体DNA分子:通过亲本DNA的随机片段化的体外同源重组,然后使用PCR进行重新装配,从而产生随机引入的点突变。使用亲本DNA分子的家族(例如来自不同物种的等位基因变体或DNA分子),可以改进这种技术,从而将其它的可变性引入该方法。选择或筛选所需活性,随后再进行另外重复的诱变和测定,通过选择所需突变并同时针对有害变化进行选择,提供序列的快速“进化”。
可将上面公开的诱变方法和高通量筛选方法相组合,以在宿主细胞中检测克隆的、诱变处理的受体的活性。在这点上优选的测定法包括细胞增殖测定法和基于生物传感器的配体结合测定法,它们描述在下面。使用现代化设备,可从宿主细胞回收诱变处理的DNA分子(其编码有活性的受体或细胞因子变体),并对其快速测序。这些方法使得能够快速确定目的多肽中的单个氨基酸残基的重要性,并可应用于未知结构的多肽。
如前所述,根据本发明的多肽融合体可以包括与特定细胞因子或细胞表面受体的细胞外结构域的“功能片段”相对应的多肽区段。可进行核酸分子的常规缺失分析,以获得编码给定的细胞因子或细胞表面受体的细胞外结构域的核酸分子的功能片段。作为说明,可用Bal31核酸酶消化具有SEQ ID NO:1的残基73-423的核苷酸序列的编码VSTM3的DNA分子,以获得一系列嵌套缺失。然后将所述片段插入表达载体的适当读码框中,并分离表达的多肽,检测其结合CD155或CD112的能力。外切核酸酶消化的一个替代方案是,使用寡核苷酸-指导的诱变以引入缺失或终止密码子,从而确定所需片段的产生。可替换地,可使用聚合酶链式反应合成编码细胞因子或受体的基因的特定片段。
通过关于在干扰素的任一端或两端处的截短的研究,举例说明这个一般性方案(参见Horisberger和Di Marco, Pharmac. Ther. 66:507, 1995)。此外,用于蛋白质的功能分析的标准技术,参见:例如,Treuter等人, Molec. Gen. Genet. 240:113, 1993;Content等人, “Expression and preliminary deletion analysis of the 42 kDa 2-5Asynthetase induced by human interferon,”Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems 65-72 (Cantell编,Nijhoff 1987);Herschman, “The EGF Receptor,”Control of Animal Cell Proliferation, 第1卷169-199 (Boynton等人编, Academic Press 1985);Coumailleau等人, J. Biol. Chem. 270:29270, 1995; Fukunaga等人, J. Biol. Chem. 270:25291,1995; Yamaguchi等人, Biochem. Pharmacol. 50:1295, 1995;和Meisel等人, Plant Molec. Biol. 30:1, 1996。
使用上面讨论的方法,本领域普通技术人员可以制备多种多肽,所述多肽:(i) 与参照多肽实质上相同,所述参照多肽与可溶性细胞因子或细胞表面受体的细胞外结构域相对应,和(ii) 保留所述参照多肽的功能性结合性质。用于确定细胞因子或受体多肽的受体或配体结合性质的测定系统,通常是本领域已知的,且可容易地改进用于确定式P1-L1-D-L2-P2和P2-L2-P1-L1-D的可溶性多肽融合体的功能性结合性质,其中P1和/或P2与细胞因子或细胞表面受体变体相对应。本文进一步描述了示例性的测定。
例如,一种优选的测定系统采用商购可得的生物传感器仪器(BIAcoreTM,Pharmacia Biosensor, Piscataway, NJ),其中将受体多肽固定化在受体芯片的表面上。Karlsson (J. Immunol. Methods 145:229-240, 1991)以及Cunningham和Wells (J. Mol. Biol. 234:554-563, 1993)公开了该仪器的用途。对于根据本发明的用途,使用胺或巯基化学法,使根据本发明的可溶性的多肽融合体(例如,可溶性的VSTM3多肽融合体) 与葡聚糖纤维共价地结合,所述葡聚糖纤维与在流动池内的金膜相连。使试验样品穿过该池。如果配体(例如,就可溶性的VSTM3多肽融合体而言,可溶性的CD155或CD112)存在于所述样品中,它将会结合固定化的多肽融合体,从而造成介质的折射率的变化,所述变化被检测为金膜的表面等离子体共振的变化。该系统允许确定结合速率(on-rate)和解离速率(off-rate),由此可以计算出结合亲和力,并评估结合的化学计量学。
所述可溶性多肽融合体还可以用在本领域已知的其它测定系统中。这样的系统包括:用于确定结合亲和力的Scatchard分析(参见Scatchard, Ann. NY Acad. Sci. 51:660-672, 1949),和测热测定法(参见Cunningham等人,Science 253:545-548, 1991;Cunningham等人, Science 254:821-825, 1991)。
在某些实施方案中,P1和P2源自细胞表面受体VSTM3 (B7R1)的细胞外结构域。因此,在有些实施方案中,根据本发明的可溶性多肽融合体从氨基端至羧基端包含选自P1-L1-D-L2-P2和P2-L2-P1-L1-D的式,其中P1是与VSTM3多肽的细胞外结构域具有至少80%序列同一性的第一种多肽,或与VSTM3细胞外结构域的功能片段具有至少80%序列同一性的第一种多肽;L1是第一种多肽接头;D是二聚化结构域;L2是第二种多肽接头;且P2是与VSTM3多肽的细胞外结构域具有至少80%序列同一性的第二种多肽,或与VSTM3细胞外结构域的功能片段具有至少80%序列同一性的第二种多肽;其中所述多肽融合体能够特异性地结合CD155多肽的细胞外结构域(例如,SEQ ID NO:22的氨基酸残基28-343或SEQ ID NO:24的氨基酸残基29-345)。在某些实施方案中,P1和/或P2与VSTM3多肽的细胞外结构域或其功能片段具有至少90%或至少95%序列同一性。例如,在有些变化中,P1和/或P2与VSTM3多肽的细胞外结构域或其功能片段具有100%序列同一性。P1和/或P2可以源自,例如,人或鼠VSTM3多肽的细胞外结构域(例如,SEQ ID NO:2的残基22-141或SEQ ID NO:4的残基26-138),或它们的功能片段。
例如,在某些实施方案中,P1和/或P2与SEQ ID NO:2的氨基酸残基25-141具有至少80%、至少90%或至少95%同一性;或P1和/或P2与SEQ ID NO:4的氨基酸残基26-138具有至少80%、至少90%或至少95%同一性。在有些这样的变化中,P1和P2中的一种或两种与SEQ IDNO:2的氨基酸残基25-141具有100%同一性,或P1和P2中的一种或两种与SEQ ID NO:4的氨基酸残基26-138具有100%同一性。在其它变化中,P1和P2中的一种或两种包含非半胱氨酸残基(例如,酪氨酸),所述非半胱氨酸残基在与SEQ ID NO:2的残基69相对应的氨基酸位置处。特别合适的P1和/或P2多肽具有在SEQ ID NO:18的残基23-139(SEQ ID NO:20的残基23-139)中所示的氨基酸序列。
在其它实施方案中,P1和/或P2与SEQ ID NO:18的氨基酸残基23-139 (SEQ IDNO:20的残基23-139)具有至少80%、至少90%或至少95%同一性。在有些这样的变化中,P1和/或P2具有非半胱氨酸残基,所述非半胱氨酸残基在与SEQ ID NO:18的残基67相对应的氨基酸位置处。例如,在具体变化中,与SEQ ID NO:18的氨基酸残基23-139具有至少80%、至少90%或至少95%同一性的P1和/或P2多肽保留与SEQ ID NO:18的酪氨酸67相对应的酪氨酸残基。
通过常规方法确定序列同一性百分比。参见,例如,Altschul等人, Bull. Math. Bio. 48:603, 1986,和Henikoff和Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1992。例如,通过使用值为10的缺口开放罚分、值为1的缺口延伸罚分和表2(氨基酸以标准的单字母代码表示)中所示的Henikoff和Henikoff(出处同上) 的“BLOSUM62”评分矩阵,可以比对2个氨基酸序列,以最优化比对评分。然后将同一性百分比计算为:([相同匹配的总数目]/[较长序列的长度+为了比对2个序列而引入较长序列中的缺口的数目])(100)。
表2:BLOSUM62评分矩阵
本领域技术人员知道,存在许多可用于比对两个氨基酸序列的已建立的算法。Pearson和Lipman的“FASTA”相似性搜索算法是适合用于检查本文公开的氨基酸序列和其假定变体的氨基酸序列所共有的同一性水平的蛋白质比对方法。所述FASTA算法参见:Pearson和Lipman, Proc. Nat’l Acad. Sci. USA 85:2444, 1988,和Pearson, Meth.Enzymol. 183:63, 1990。简而言之,FastA首先如下表征序列相似性:在不考虑保守氨基酸置换、插入或缺失的情况下,鉴定查询序列(例如,SEQ ID NO:2的残基25-141或SEQ ID NO:18的残基23-139)和测试序列共有的区域,所述区域具有最高同一性密度(如果ktup变量=1)或成对同一性(如果ktup=2)。然后如下给具有最高同一性密度的10个区域重新评分:使用氨基酸置换矩阵,比较所有成对氨基酸的相似性,“修剪”所述区域的末端,以仅包括对最高分值有贡献的那些残基。如果存在几个具有高于“截止”值的评分(基于序列长度和ktup值,通过预设的公式计算)的区域,那么检查经修剪的起始区域,以确定是否可连接所述区域以形成具有缺口的近似比对。最后,使用允许氨基酸插入和缺失的Needleman-Wunsch-Sellers算法(Needleman和Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J.Appl. Math. 26:787, 1974)的修改方案,对两个氨基酸序列的最高评分区域进行比对。FASTA分析的示例性参数是:ktup=1、缺口开放罚分=10、缺口延伸罚分=1、和取代矩阵=blosum62。如在Pearson, Meth. Enzymol. 183:63, 1990的附录2中所解释的,通过修改评分矩阵文件(“SMATRIX”),可将这些参数引入FASTA程序中。
也可利用如上文公开的比值,使用FASTA确定核酸分子的序列同一性。对于核苷酸序列的比较,ktup值可在1至6之间、优选地从3至6的范围内变化,最优选3,其它参数如上设定。
本发明包括可溶性VSTM3多肽融合体,其与SEQ ID NO:2的残基25-141或SEQ IDNO:18的残基23-139 (SEQ ID NO:20的残基23-139)的氨基酸序列相比,具有保守性氨基酸改变。例如,可获得含有SEQ ID NO:2的残基25-141或SEQ ID NO:18的残基23-139中的一个或多个氨基酸置换的VSTM3变体,其中用烷基氨基酸置换在VSTM3氨基酸序列中的烷基氨基酸,用芳族氨基酸置换在VSTM3氨基酸序列中的芳族氨基酸,用含硫氨基酸置换在VSTM3氨基酸序列中的含硫氨基酸,用含羟基的氨基酸置换在VSTM3氨基酸序列中的含羟基的氨基酸,用酸性氨基酸置换在VSTM3氨基酸序列中的酸性氨基酸,用碱性氨基酸置换在VSTM3氨基酸序列中的碱性氨基酸,或用二碱式一羧基氨基酸(dibasic monocarboxylic aminoacid)置换在VSTM3氨基酸序列中的二碱式一羧基氨基酸氨基酸。在常见氨基酸中,通过例如在下列每个组内的氨基酸之间的置换来举例说明“保守氨基酸置换”:(1)甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸,(2) 苯丙氨酸、酪氨酸和色氨酸,(3)丝氨酸和苏氨酸,(4)天冬氨酸和谷氨酸,(5)谷氨酰胺和天冬酰胺,和(6)赖氨酸、精氨酸和组氨酸。保守性氨基酸改变的示例组进一步显示在上述文献的表1中。
BLOSUM62表(参见表2)是氨基酸置换矩阵,其来源于代表超过500组关联蛋白的高度保守区域的蛋白质序列区段的约2,000个局部多重比对(Henikoff和Henikoff, Proc.Nat’l Acad. Sci. USA 89:10915, 1992)。因此,可以使用BLOSUM62置换频率来限定可被引入本发明的氨基酸序列中的保守氨基酸置换。尽管可能只基于化学性质(如上所述)来设计氨基酸置换,描述“保守氨基酸置换”优选指由大于-1的BLOSUM62值代表的置换。例如,如果置换的特征在于0、1、2或3的BLOSUM62值,那么该氨基酸置换是保守的。根据该系统,优选的保守氨基酸置换的特征在于至少1 (例如,1、2或3)的BLOSUM62值,而更优选的保守氨基酸置换的特征在于至少2 (例如,2或3)的BLOSUM62值。细胞因子或受体多肽(例如,VSTM3)的特定变体的特征在于,其与对应的氨基酸序列(例如,SEQ ID NO:2的残基25-141或SEQID NO:18的残基23-139)具有至少80%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性,其中氨基酸序列中的变异是由于一个或多个保守氨基酸置换。
多种二聚化结构域适合用于本文所述的二聚融合蛋白中。在某些实施方案中,所述二聚化结构域是免疫球蛋白重链恒定区,诸如Fc区。所述Fc区可以是天然序列Fc区或变体Fc区。在有些实施方案中,所述Fc区缺少一种或多种效应物功能(例如,ADCC和CDC效应物功能中的一种或两种)。缺少一种或多种效应物功能的示例性Fc区包括,例如,Fc-488、Fc4、Fc5、Fc6和Fc7 (参见图2A-2C;SEQ ID NO:29-33,分别从氨基酸残基16开始)。
根据本发明使用的多肽接头可以是天然存在的、合成的或二者的组合。所述接头连接2个单独的多肽区域(例如,二聚化结构域和与VSTM3 (B7R1)的细胞外结构域相对应的多肽),并维持所述连接的多肽区域作为更长多肽的单独的且不连续的结构域。所述接头可以使单独的、不连续的结构域协作,且仍然维持各自的性质(例如,就与VSTM3的细胞外结构域的对应多肽相连的Fc区二聚化结构域而言,可以维持所述Fc区与Fc受体(例如,FcRn)的结合,同时维持VSTM3细胞外结构域的CD155结合性质)。天然存在的以及人工的肽接头用于将多肽连接成新的连接的融合多肽的用途,是本领域众所周知(参见,例如,Hallewell等人,J. Biol. Chem. 264, 5260-5268, 1989; Alfthan等人,Protein Eng. 8, 725-731,1995; Robinson和Sauer, Biochemistry 35, 109-116, 1996; Khandekar等人,J. Biol. Chem. 272, 32190-32197, 1997; Fares等人,Endocrinology 139, 2459-2464, 1998;Smallshaw等人,Protein Eng. 12, 623-630, 1999;美国专利号5,856,456)。
通常,选择在接头多肽内的残基,以提供总的亲水特性,并且是非免疫原性的和柔性的。本文使用的“柔性的”接头是这样的接头:其在溶液中缺少实质上稳定的有序性更高的构象,尽管局部稳定性区域是可允许的。一般而言,较小的、极性的和亲水的残基是优选的,大体积的和疏水的残基是不希望的。应当避免局部电荷区域;如果接头多肽包括带电荷的残基,它们通常如此定位:以便在所述多肽的小区域内提供净中性电荷。因此,优选的是,将带电荷的残基放在具有相反电荷的残基附近。一般而言,在接头多肽内包含的优选残基包括:Gly、Ser、Ala、Thr、Asn和Gln;更优选的残基包括:Gly、Ser、Ala和Thr;且最优选的残基是Gly和Ser。一般而言,应当避免Phe、Tyr、Trp、Pro、Leu、Ile、Lys和Arg残基(除非存在于接头的免疫球蛋白铰链区内),Pro残基是由于它们的疏水性和缺乏柔性,Lys和Arg残基是由于潜在的免疫原性。如本文公开的,将包括Cys残基,以便提供二硫键合。还设计接头的序列,以避免不希望的蛋白酶解。
在有些实施方案中,所述多肽接头L1包含15-32个氨基酸残基或由它们组成,其中1-8个所述残基是半胱氨酸残基。在本发明的一个优选实施方案中,每个接头精确地含有2个半胱氨酸残基。设计所述接头,以在二聚化结构域和P1多肽(例如,与VSTM3细胞外结构域相对应的P1多肽)之间提供足够的空间和柔性,从而允许所述结构域在所述多肽内行使它们的预期功能。选择接头长度和组成,以提供期望的间距和柔性程度,同时也提供一个或更多个链间二硫键,以稳定期望的构象。
在具体变化中,L1是免疫球蛋白铰链区,或免疫球蛋白铰链区的片段或变体。在本发明的一个实施方案中,通过用另一种氨基酸残基(例如,Ser)替代,或通过缺失或截短,从铰链中省略最靠近N-端的半胱氨酸残基(Eu残基220;SEQ ID NO:27的残基103),所述半胱氨酸残基在装配好的天然抗体中与免疫球蛋白轻链形成二硫键。还可以做出在铰链序列中的其它变化。例如,可以将Lys残基(Eu 218;SEQ ID NO:27的残基101) 改成Arg。所述多肽接头因而可以包含含有至少2个半胱氨酸残基的免疫球蛋白铰链区或其片段或变体,所述半胱氨酸残基与其它链上的多肽接头形成二硫键。从任意免疫球蛋白重链可以得到免疫球蛋白铰链区。已经确定地表征了γ(IgG) 铰链区,诸如γ1铰链,且方便地用于本发明中。
示例性的L2多肽接头包含多个甘氨酸残基。例如,在有些实施方案中,L2多肽接头包含多个甘氨酸残基和任选的至少一个丝氨酸残基。在包含式P1-L1-D-L2-P2的多肽的具体变化中,L2包含式Gly-Gly-Gly-Ser-Gly (SEQ ID NO:21)。在包含式P2-L2-P1-L1-D的多肽的具体变化中,L2多肽接头包含式[Gly-Gly-Gly-Ser]n (SEQ ID NO:22),其中n是3-5的整数。在包含式[Gly-Gly-Gly-Ser]n的L2接头的一个具体变化中,n是4。
用于本发明内的多肽区段(例如,与VSTM3细胞外结构域相对应的多肽区段,包含免疫球蛋白铰链区的接头,和二聚化结构域诸如Fc片段),可以从多种物种得到。如果要在人类中治疗性地使用所述二聚蛋白,优选地采用人多肽序列。但是,可以使用非人序列,也可以使用变体序列。对于其它应用,包括体外诊断应用和兽医学应用,可以采用得自人类或非人动物的多肽序列,尽管得自与患者相同物种的序列就体内兽医学应用而言或就体外应用(其中存在分子间反应的物种特异性)而言可能是优选的。因而,用于本发明内的多肽区段可以是,但不限于,人、非人灵长类动物、啮齿动物、犬科动物、猫科动物、马科动物、牛科动物、羊、猪、兔类动物和禽类的多肽以及它们的变体。
在包含式P1-L1-D-L2-P2(且其中P1和P2中的每一种源自VSTM3 (B7R1)的细胞外结构域)的多肽融合体的具体实施方案中,所述多肽融合体包含在下述序列中所示的氨基酸序列:SEQ ID NO:18的残基23-493或1-493;SEQ ID NO:6的残基22-498或1-498;SEQ IDNO:10的残基26-489或1-489,SEQ ID NO:14的残基36-506或1-506,SEQ ID NO:16的残基36-506或1-506,或SEQ ID NO:18的残基23-493或1-493。在包含式P2-L2-P1-L1-D(且其中P1和P2中的每一种源自VSTM3的细胞外结构域)的多肽融合体的具体实施方案中,所述多肽融合体包含在下述序列中所示的氨基酸序列:SEQ ID NO:20的残基23-508、23-507、1-508或1-507;SEQ ID NO:8的残基22-513、22-512、1-513或1-512;SEQ ID NO:12的残基26-504、26-503、1-504或1-503;或SEQ ID NO:20的残基23-508、23-507、1-508或1-507。
本发明也提供了二聚蛋白,其包含如上所述的第一种和第二种多肽融合体。因此,在另一个方面,本发明提供了包含第一种多肽融合体和第二种多肽融合体的二聚蛋白,其中所述第一种和第二种多肽融合体中的每一种从氨基端至羧基端包含本文所述的P1-L1-D1-L2-P2或P2-L2-P1-L1-D。例如,在具体实施方案中,根据本发明的二聚的VSTM3 (B7R1)蛋白包含第一种多肽融合体和第二种多肽融合体,其中所述第一种和第二种多肽融合体中的每一种从氨基端至羧基端包含P1-L1-D1-L2-P2,其中P1和P2中的每一种源自VSTM3的细胞外结构域,且其中所述二聚蛋白能够特异性地结合CD155的细胞外结构域(例如,SEQ IDNO:22的氨基酸残基28-343)。在其它实施方案中,根据本发明的二聚的VSTM3蛋白包含第一种多肽融合体和第二种多肽融合体,其中所述第一种和第二种多肽融合体中的每一种从氨基端至羧基端包含P2-L2-P1-L1-D,其中P1和P2中的每一种源自VSTM3的细胞外结构域,且其中所述二聚蛋白能够特异性地结合CD155的细胞外结构域。
III. 用于制备多肽融合体和二聚蛋白的材料和方法
本发明也提供了编码上面公开的融合多肽的多核苷酸分子,包括DNA和RNA分子。本发明的多核苷酸包括单链和双链分子。使用已知的核酸重组操作方法,可以制备编码多肽融合体的不同区段(例如,二聚化结构域诸如Fc片段;P1和P2多肽区段) 的多核苷酸,并连接到一起以形成编码本文所述的多肽融合体的多核苷酸。
编码细胞因子和细胞表面受体(例如,VSTM3 (B7R1)),包括与这样的受体的细胞外结构域相对应的多肽区段)的DNA序列是本领域已知的。编码各种二聚化结构域(例如,免疫球蛋白重链恒定区诸如Fc片段) 的DNA序列也是已知的。基于遗传密码,本领域普通技术人员可以容易地制备编码细胞因子、细胞表面受体和二聚化结构域多肽的其它DNA序列。通过用U置换T,可以制备对应的RNA序列。本领域技术人员可容易地认识到,考虑到遗传密码的简并性,在编码给定多肽的多核苷酸分子中的大量序列变异是可能的。还可以如下得到编码这种多肽的功能变体和片段的DNA和RNA:使用已知的重组方法将变异引入多核苷酸序列中,随后表达编码的多肽,并使用适当的筛选测定法确定功能活性(例如,与相应受体或配体的结合)。
用于制备DNA和RNA的方法是本领域众所周知的。例如,可以从分离自组织或细胞的RNA制备互补DNA (cDNA) 克隆,所述组织或细胞生产大量编码目标多肽的RNA。可以如下制备总RNA:使用盐酸胍提取,随后在CsCl梯度中离心分离(Chirgwin等人, Biochemistry18:52-94, 1979)。使用Aviv和Leder的方法(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972),从总RNA制备聚(A)+ RNA。使用已知方法,从聚(A)+ RNA制备互补DNA。在替代方案中,可以分离基因组DNA。对于某些用途(例如,在转基因动物中表达),可能有利的是,使用基因组克隆,或修饰cDNA克隆以包括至少一个基因组内含子。用于鉴定和分离cDNA和基因组克隆的方法是众所周知的,且在本领域的普通技术水平内,包括本文公开的序列或其部分用于探测或引发文库的用途。通过例如杂交或聚合酶链式反应(“PCR,”Mullis, 美国专利4,683,202),鉴定和分离编码目标多肽的多核苷酸。可以用针对目标多肽、受体片段或其它特异性的结合伴侣的抗体探测表达文库。
也可以通过自动化合成来制备本发明的多核苷酸。短的双链区段(60-80 bp)的生产在技术上是简单的,且可以如下实现:合成互补链,然后使它们退火。从长度为20-100个核苷酸的单链片段,以模块形式装配更长的区段(通常>300 bp)。多核苷酸的自动化合成是在本领域的普通技术水平内,合适的设备和试剂可从商业供应商得到。一般参见Glick和Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura等人,Ann. Rev. Biochem. 53:323-356, 1984;和Climie等人,Proc. Natl. Acad. Sci. USA 87:633-637, 1990。
在另一个方面,提供了用于生产本发明的多肽融合体(包括含有所述多肽融合体的二聚蛋白)的材料和方法。根据常规技术,可以在遗传工程改造的宿主细胞中生产所述多肽融合体。合适的宿主细胞是可以用外源DNA转化或转染并在培养物中生长的那些细胞类型,包括细菌、真菌细胞和培养的高等真核细胞(包括培养的多细胞生物体的细胞),特别是培养的哺乳动物细胞。用于操作克隆的DNA分子和将外源DNA引入多种宿主细胞中的技术,参见:Sambrook等人, Molecular Cloning:A Laboratory Manual, 第2版, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989,和Ausubel等人编,Current Protocols in Molecular Biology, Green and Wiley and Sons, NY, 1993。
一般而言,将编码多肽融合体的DNA序列可操作地连接至在表达载体内的其表达所需的其它基因元件上,通常包括转录启动子和终止子。所述载体一般也含有一种或多种选择标记和一个或多个复制起点,尽管本领域技术人员会认识到,在某些系统内,可在分开的载体上提供选择标记,和通过将外源DNA整合进宿主细胞基因组中来提供所述外源DNA的复制。启动子、终止子、选择标记、载体和其它元件的选择,是在本领域普通技术水平之内的常规设计的内容。在文献中描述了许多这样的元件,并且可从商业供应商获得。
为了引导多肽融合体进入宿主细胞的分泌途径中,在表达载体中提供分泌信号序列。所述分泌信号序列可以是天然的非免疫球蛋白多肽的分泌信号序列,或者可以源自其它分泌型蛋白(例如,t-PA;参见美国专利号5,641,655),或从头合成。可以在分泌肽与多肽融合体的其它部分之间的接合处包括工程改造的切割位点,以优化在宿主细胞中的蛋白水解加工。所述分泌信号序列与编码多肽融合体的DNA序列可操作地相连,即,2个序列连接在正确的读码框中,且定位成引导新合成的多肽融合体进入宿主细胞的分泌途径中。分泌信号序列通常位于编码目标多肽的DNA序列的5'侧,尽管某些信号序列可以位于目标DNA序列中的别处(参见,例如,Welch等人, 美国专利号5,037,743; Holland等人, 美国专利号5,143,830)。适合用于根据本发明的用途的分泌信号序列包括,例如,编码SEQ ID NO:14的氨基酸残基1-35或SEQ ID NO:18的氨基酸残基1-22的多核苷酸。
预期通过宿主细胞分泌途径的多肽融合体的表达会导致二聚蛋白的生成。因此,在另一个方面,本发明提供了包含如上所述的第一种和第二种多肽融合体的二聚蛋白(例如,包含第一种多肽融合体和第二种多肽融合体的二聚蛋白,其中所述第一种和第二种多肽融合体中的每一种从氨基端至羧基端包含P1-L1-D1-L2-P2,或其中所述第一种和第二种多肽融合体中的每一种从氨基端至羧基端包含P2-L2-P1-L1-D,且其中所述二聚蛋白能够特异性地结合CD155的细胞外结构域(例如,SEQ ID NO:22的氨基酸残基28-343))。在合适的条件下孵育组分多肽以后,也可以在体外装配二聚体。一般而言,体外装配包括:在变性和还原条件下孵育蛋白混合物,随后重新折叠和重新氧化所述多肽,以形成二聚体。下面公开了在细菌细胞中表达的蛋白的回收和装配。
培养的哺乳动物细胞是适合用于本发明的用途的宿主。用于将外源DNA引入哺乳动物宿主细胞中的方法包括:磷酸钙介导的转染(Wigler等人,Cell 14:725, 1978;Corsaro和Pearson, Somatic Cell Genetics 7:603, 1981: Graham和Van der Eb,Virology 52:456, 1973)、电穿孔(Neumann等人,EMBO J. 1:841-845, 1982)、DEAE-葡聚糖介导的转染(Ausubel等人, 出处同上)和脂质体介导的转染(Hawley-Nelson等人,Focus15:73, 1993; Ciccarone等人, Focus 15:80, 1993)。重组多肽在培养的哺乳动物细胞中的生产,参见:例如,Levinson等人, 美国专利号4,713,339; Hagen等人, 美国专利号4,784,950; Palmiter等人, 美国专利号4,579,821;和Ringold, 美国专利号4,656,134。合适的培养的哺乳动物细胞包括:COS-1 (ATCC No. CRL 1650)、COS-7 (ATCC No. CRL1651)、BHK (ATCC No. CRL 1632)、BHK 570 (ATCC No. CRL 10314)、293 (ATCC No. CRL1573; Graham等人, J. Gen. Virol. 36:59-72, 1977)和中国仓鼠卵巢(例如,CHO-K1,ATCC No. CCL 61; CHO-DG44, Urlaub等人,Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) 细胞系。其它合适的细胞系是本领域已知的,且可从公开的保藏机构(诸如美国典型培养物保藏中心, 马纳萨斯, 弗吉尼亚州)得到。可以使用强转录启动子,诸如得自SV-40、巨细胞病毒或骨髓增生性肉瘤病毒的启动子。参见,例如,美国专利号4,956,288和美国专利申请公开号20030103986。其它合适的启动子包括:得自金属硫蛋白基因的那些(美国专利号4,579,821和4,601,978),和腺病毒主要晚期启动子。用于哺乳动物细胞中的表达载体包括:pZP-1、pZP-9和pZMP21,它们已经分别在登录号98669、98668和PTA-5266下保藏在美国典型培养物保藏中心(10801 University Blvd., 马纳萨斯, 美国弗吉尼亚州),和这些载体的衍生物。
通常使用药物选择来选择其中已经插入外源DNA的培养的哺乳动物细胞。这样的细胞通常称作“转染子”。已经在有选择剂存在下培养且能够将目标基因传递给它们的后代的细胞称作“稳定的转染子”。一种示例性的选择标记是编码对抗生素新霉素的抗性的基因。在新霉素类型药物(例如G-418等)存在下进行选择。也可使用选择系统增加目标基因的表达水平,即被称为“扩增”的方法。如下进行扩增:在有低水平选择剂存在下培养转染子,随后增加选择剂的量,以选择产生高水平的被导入基因的产物的细胞。一种示例性的可扩增的选择标记是二氢叶酸还原酶,其赋予针对甲氨蝶呤的抗性。也可使用其它抗药性基因(例如,潮霉素抗性基因、广谱抗药基因、嘌呤霉素乙酰转移酶基因)。细胞表面标志物和其它表型选择标志物可以用于促进转染的细胞的鉴定(例如,通过荧光活化的细胞分选),且包括,例如,CD8、CD4、神经生长因子受体、绿色荧光蛋白等。
其它高等真核细胞也可以用作宿主,包括昆虫细胞、植物细胞和禽细胞。Sinkar等人,J. Biosci. (Bangalore) 11:47-58, 1987已经综述了毛根土壤杆菌(Agrobacterium rhizogenes)作为用于在植物细胞中表达基因的载体的用途。Guarino等人, 美国专利号5,162,222和WIPO公开WO 94/06463公开了昆虫细胞的转化和外来多肽在其中的生产。
可以用重组杆状病毒感染昆虫细胞,所述重组杆状病毒通常源自苜蓿银纹夜蛾核型多角体病毒(AcNPV)。参见King和Possee,The Baculovirus Expression System:A Laboratory Guide, Chapman & Hall, London; O'Reilly等人,Baculovirus Expression Vectors:A Laboratory Manual, Oxford University Press., New York, 1994;和 Richardson编, Baculovirus Expression Protocols. Methods in Molecular Biology,Humana Press, Totowa, NJ, 1995。通过使用Luckow等人(J. Virol. 67:4566-4579,1993)所述的基于转座子的系统,也可以生产重组杆状病毒。该系统(其使用转移载体)可以以试剂盒形式(BAC-TO-BAC试剂盒; Life Technologies, Gaithersburg, MD)商购得到。所述转移载体(例如,PFASTBAC1; Life Technologies) 含有Tn7转座子,以将编码目标蛋白的DNA转移进杆状病毒基因组中,所述杆状病毒基因组在大肠杆菌中维持为大质粒,称作“杆粒(bacmid)”。参见Hill-Perkins和Possee,J. Gen. Virol. 71:971-976, 1990;Bonning等人,J. Gen. Virol. 75:1551-1556, 1994;以及Chazenbalk和Rapoport, J. Biol. Chem. 270:1543-1549, 1995。使用本领域已知的技术,将编码多肽融合体的转移载体转化进大肠杆菌宿主细胞中,并筛选细胞中的杆粒,所述杆粒含有指示重组杆状病毒的中断的lacZ基因。使用普通技术,分离含有重组杆状病毒基因组的杆粒DNA,并用于转染草地贪夜蛾(Spodoptera frugiperda)细胞,诸如Sf9细胞。随后生产表达多肽融合体的重组病毒。通过本领域常用的方法,制备重组病毒储备物。
为了蛋白生产,使用重组病毒感染宿主细胞,通常是源自秋粘虫草地贪夜蛾(例如,Sf9或Sf21细胞)或粉纹夜蛾(Trichoplusia ni)(例如,HIGH FIVE细胞; Invitrogen,Carlsbad, CA)的细胞系。一般参见Glick和Pasternak, 出处同上。也参见美国专利号5,300,435。使用无血清培养基来培养和维持细胞。合适的培养基制剂是本领域已知的,且可以从商业供应商得到。使细胞从约2-5 x 105细胞的接种密度生长至1-2 x 106细胞的密度,在此时以0.1-10(更典型地,接近3)的感染复数(MOI)加入重组病毒储备物。使用的操作通常参见可得到的实验室手册(例如,King和Possee,出处同上; O'Reilly等人, 出处同上.;Richardson, 出处同上)。
在本发明中也可使用真菌细胞,包含酵母细胞。在这点上,特别感兴趣的酵母物种包括:酿酒酵母、巴氏毕赤酵母和甲醇毕赤酵母。用外源DNA转化酿酒酵母细胞和由此生产重组多肽的方法,参见:例如,Kawasaki, 美国专利号4,599,311; Kawasaki等人, 美国专利号4,931,373; Brake, 美国专利号4,870,008; Welch等人, 美国专利号5,037,743;和Murray等人, 美国专利号4,845,075。通过由选择标记确定的表型,通常为抗药性或在特定营养物(例如,亮氨酸)不存在的情况下的生长能力,选择转化的细胞。在酿酒酵母中使用的一种示例性的载体系统是由Kawasaki等人(美国专利号4,931,373)公开的POT1载体系统,该系统允许通过在含有葡萄糖的培养基中的生长来选择转化的细胞。在酵母中使用的其它合适的启动子和终止子包括来自糖酵解酶基因(参见,例如,Kawasaki, 美国专利号4,599,311; Kingsman等人, 美国专利号4,615,974;和Bitter, 美国专利号4,977,092)和醇脱氢酶基因的启动子和终止子。也参见美国专利号4,990,446、5,063,154、5,139,936和4,661,454。用于其它酵母的转化系统是本领域已知的,所述其它酵母包括:多形汉逊酵母、粟酒裂殖酵母、乳酸克鲁维酵母、脆壁克鲁维酵母、玉蜀黍黑粉菌、巴氏毕赤酵母、甲醇毕赤酵母、季也蒙氏毕赤酵母(Pichia guillermondii)和麦芽糖假丝酵母(Candida maltosa)。参见,例如,Gleeson等人,J. Gen. Microbiol. 132:3459-3465, 1986; Cregg, 美国专利号4,882,279;和Raymond等人, Yeast 14:11-23, 1998。根据McKnight等人, 美国专利号4,935,349的方法,可以使用曲霉菌细胞。Sumino等人, 美国专利号5,162,228公开了转化产黄支顶孢(Acremonium chrysogenum)的方法。Lambowitz, 美国专利号4,486,533公开了转化链孢霉(Neurospora)的方法。美国专利号5,716,808、5,736,383、5,854,039和5,888,768公开了重组蛋白在甲醇毕赤酵母中的生产。
原核宿主细胞,包括细菌大肠杆菌、芽孢杆菌属和其它属的菌株,也是在本发明中有用的宿主细胞。用于转化这些宿主和表达在其中克隆的外源DNA序列的技术,是本领域众所周知的(参见,例如,Sambrook等人, 出处同上)。当在细菌(例如大肠杆菌)中表达多肽融合体时,所述多肽可保留在细胞质中,通常作为不溶性颗粒,或可被细菌分泌序列引导至周质间隙。在前一种情况下,裂解细胞,回收所述颗粒,并使用例如盐酸胍或尿素使之变性。然后可以如下使变性的多肽重折叠和二聚化:稀释变性剂,例如通过在尿素溶液以及还原型和氧化型谷胱甘肽的组合中透析,然后在缓冲盐水溶液中透析。在替代方案中,以可溶形式从细胞质中回收所述蛋白,并且不使用变性剂进行分离。从细胞回收蛋白,作为在例如磷酸盐缓冲盐水中的水性提取物。为了捕获目标蛋白,将所述提取物直接应用于色谱介质,诸如固定化的抗体或肝素-琼脂糖柱。可如下以可溶性和功能性的形式从周质间隙回收分泌的多肽:破裂细胞(通过例如超声处理或渗透冲击),并回收所述蛋白质,从而避免对变性和重折叠的需要。参见,例如,Lu等人, J. Immunol. Meth. 267:213-226, 2002。
根据常规操作,在培养基中培养转化的或转染的宿主细胞,所述培养基含有营养物和选择的宿主细胞的生长所需的其它组分。多种合适的培养基(包括确定成分培养基和复合培养基)是本领域已知的,且通常包括碳源、氮源、必需氨基酸、维生素和矿物质。培养基也可以含有诸如生长因子或血清等组分,视需要而定。生长培养基通常通过例如药物选择或必需营养物的缺乏(其被表达载体携带的或共转染进宿主细胞中的选择标记补足)来选择含有外源地添加的DNA的细胞。
通过常规蛋白纯化方法,通常通过色谱技术的组合,纯化本发明的蛋白。一般参见Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, 瑞典, 1988;和Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994。通过在固定化的蛋白A上的亲和色谱法,可以纯化包含免疫球蛋白重链多肽的蛋白。可以使用其它纯化步骤(诸如凝胶过滤)得到期望的纯度水平,或提供用于脱盐、缓冲液更换等。
例如,可以使用分级分离和/或常规纯化方法得到从重组宿主细胞中纯化出的本发明的多肽融合体和二聚蛋白。一般而言,可以使用硫酸铵沉淀法和酸或离液剂提取法,进行样品的分级分离。示例性的纯化步骤可以包括:羟磷灰石、大小排阻、FPLC和反相高效液相色谱法。合适的色谱介质包括:衍生化的葡聚糖、琼脂糖、纤维素、聚丙烯酰胺、特种硅土(specialty silicas)等。PEI、DEAE、QAE和Q衍生物是合适的。示例性的色谱介质包括:用苯基、丁基或辛基衍生化的那些介质,例如苯基-琼脂糖FF(Pharmacia)、Toyopearl丁基650(Toso Haas, Montgomeryville, PA)、辛基-琼脂糖(Pharmacia)等;或聚丙烯酸树脂,例如Amberchrom CG 71 (Toso Haas) 等。合适的固体支持物包括玻璃珠粒、基于二氧化硅的树脂、纤维素树脂、琼脂糖珠粒、交联的琼脂糖珠粒、聚苯乙烯珠粒、交联的聚丙烯酰胺树脂等,所述支持物在它们使用条件下是不溶性的。可以用反应基团修饰这些支持物,所述反应基团允许通过氨基、羧基、巯基、羟基和/或糖部分连接蛋白质。
偶联化学试剂的实例包括:溴化氰活化、N-羟基琥珀酰亚胺活化、环氧化物活化、巯基活化、酰肼活化和用于碳二亚胺偶联化学法的羧基和氨基衍生物。这些和其它固体介质是本领域内熟知的和广泛使用的,并且可从商业供应商获得。用于多肽分离和纯化的特定方法的选择,是在常规设计的内容,并且部分地取决于选择的支持物的特性。参见,例如,Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology1988);和Doonan, Protein Purification Protocols (The Humana Press 1996)。
本领域技术人员可以设计在蛋白分离和纯化中的其它变化。例如,可以使用特异性地结合本文所述的多肽融合体或二聚蛋白的抗体(例如,特异性地结合与细胞因子或细胞表面受体的细胞外结构域相对应的多肽区段的抗体)通过免疫亲和纯化来分离大量蛋白。
通过利用特定性质,也可分离本发明的蛋白。例如,可使用固定化的金属离子吸附(IMAC)色谱法纯化富含组氨酸的蛋白质,包括含有多组氨酸标签的蛋白质。简而言之,首先用二价金属离子加载凝胶以形成螯合物(Sulkowski, Trends in Biochem. 3:1, 1985)。依赖于所用的金属离子,可将富含组氨酸的蛋白质吸附至具有不同亲和力的这种基质上,并通过竞争性洗脱、降低pH、或使用强螯合剂,洗脱所述蛋白。其它纯化方法包括:通过凝集素亲和色谱法和离子交换色谱法,纯化糖基化的蛋白(参见,例如,M. Deutscher, (编),Meth. Enzymol. 182:529, 1990)。在本发明的其它实施方案中,可构建目标多肽和亲和标签(例如,麦芽糖结合蛋白、免疫球蛋白结构域)的融合体,以促进纯化。此外,可使用二聚体蛋白的受体或配体结合性质进行纯化。例如,可以使用亲和色谱法分离二聚的VSTM3蛋白,在所述亲和色谱法中,将CD155结合至柱上,结合二聚的VSTM3蛋白,随后使用标准的色谱方法洗脱。
相对于污染性大分子(特别是其它蛋白质和核酸),通常将本发明的多肽纯化至至少约80% 纯度,更通常至至少约90% 纯度,优选地至至少约95%、至少约96%、至少约97%、至少约98%或至少约99% 纯度,并且不含感染性因子和致热性因子。也可将本发明的多肽纯化至药物纯度状态,这是大于99.9%的纯度。在某些制剂中,纯化的多肽基本上不含其它多肽,特别是动物来源的其它多肽。
IV. 使用方法和药物组合物
本发明的二聚蛋白可以用于诊断、治疗或研究,以提供一种或多种与P1和P2多肽有关的活性。这样的活性包括、但不限于:受体结合、受体活化和配体结合。本领域技术人员可容易地预见到所述蛋白的应用范围。治疗用途包括,例如,用作细胞因子拮抗剂,诸如用于治疗癌症或免疫学障碍,和用作生长因子激动剂,诸如用于促进组织生长或愈合,或促进脉管系统或其它组织的发育。诊断用途包括,例如,用作放射性同位素或其它标记的靶向剂,用于检测分子在细胞表面上或在生物流体或提取物中的存在,或用作体外测定中的对照。在研究中,本发明的蛋白可以用于例如标记细胞,从而测定细胞表面受体或可溶性分子的存在,和用于研究细胞因子或受体多肽或它们的结合伴侣的生物学。
在一个具体方面,本发明提供了治疗T-细胞介导的免疫障碍的方法。所述方法通常包括:给具有T-细胞介导的免疫障碍的受试者施用有效量的本文所述的二聚的VSTM3(B7R1) 蛋白。适合根据本发明的治疗的T-细胞介导的免疫障碍包括,例如,自身免疫疾病、移植物抗宿主病(GVHD)和移植排斥。T-细胞介导的自身免疫疾病的实例包括:类风湿性关节炎、多发性硬化(MS) (例如,脊髓-视觉多发性硬化、原发进行性多发性硬化(PPMS)和复发缓解型多发性硬化(RRMS))、胰岛素依赖性的糖尿病(IDDM)、系统性红斑狼疮(SLE)、腹部疾病、神经炎、多肌炎、银屑病、银屑病关节炎、白癫风、干燥综合征、自身免疫胰腺炎、炎性肠病(例如,克罗恩氏病、溃疡性结肠炎)、活动性慢性肝炎、肾小球肾炎、硬皮病、结节病、自身免疫甲状腺疾病、桥本甲状腺炎、格雷夫斯病、韦格纳氏肉芽肿病、重症肌无力、哮喘、阿狄森氏病、自身免疫性葡萄膜炎、寻常型天疱疮、原发性胆汁性肝硬化、恶性贫血、交感性眼炎、葡萄膜炎、自身免疫性溶血性贫血、肺纤维化、慢性铍病和特发性肺纤维化,仅举几个例子。
就治疗用途而言,本文所述的二聚蛋白的递送方式与要治疗的疾病或障碍的处置相关的常规方法一致。按照本文的公开内容,将有效量的二聚蛋白施用给需要这类治疗的受试者,并且施用时间和条件足以预防或治疗所述疾病或障碍。
被施用本文描述的二聚蛋白的受试者包括:处于发展特定疾病或障碍的高风险中的患者,以及表现出现有疾病或障碍的患者。在某些实施方案中,受试者已被诊断为患有需要治疗的疾病或障碍。此外,在治疗过程中可以监测受试者的疾病或障碍的任何变化(例如疾病或障碍的临床症状增加或减少)。并且,在有些变化中,所述受试者没有罹患需要下述治疗的其它疾病或障碍:所述治疗涉及施用与所述二聚蛋白的P1或P2多肽区段相对应的细胞因子或受体多肽。
在预防性应用中,以足够消除或减少疾病风险或者延迟疾病发作的量,将药物组合物或药物施用给患者,所述患者易患特定疾病或者以其它方式处于所述特定疾病的风险中。在治疗性应用中,以足够治愈或至少部分地遏制疾病症状及其并发症的量,将组合物或药物施用给患者,所述患者疑似或者已经罹患这类疾病。足够实现该目的的量被称作治疗有效的或药学有效的剂量或量。在预防和治疗方案中,通常以多个剂量施用药剂,直至已经达到足够的反应(例如不适当的T细胞应答的抑制)。通常,监测反应,如果期望的反应开始消退,给予重复剂量。
为了鉴定按照本发明的方法进行治疗的受试患者,可以采用获得认可的筛选方法,确定与特定疾病相关的风险因素,或确定在受试者中鉴定的现有疾病的状态。这类方法可以包括,例如,确定个体是否有亲戚已被诊断出特定疾病。筛选方法还可以包括,例如,用于确定已知具有遗传性组成的特定疾病的家族状况的常规工作。为此目的,可以常规地使用核苷酸探针来鉴定携带与特定目标疾病相关的遗传标记的个体。此外,大量用于鉴定特定疾病的标记物的免疫学方法是本领域已知的。按照已知的患者症状学、年龄因素、相关风险因素等的指示,可以实现筛选。这些方法使得临床医师能够常规地选择需要利用本文描述的方法进行治疗的患者。与这些方法相对应,使用本发明的二聚蛋白的治疗(例如,使用二聚的VSTM3蛋白来抑制不适当的T-细胞应答的治疗) 可以作为独立的治疗计划,或者作为其它治疗的随访、附加或协同治疗方案。
为了施用,将根据本发明的二聚蛋白配制成药物组合物。根据制备药学有用的组合物的已知方法,可以配制包含二聚蛋白的药物组合物,其中将治疗性分子与药学上可接受的载体组合成混合物。如果受体患者可以耐受施用,则组合物被称为“药学上可接受的载体”。无菌的磷酸盐缓冲盐水是药学上可接受的载体的一个例子。其它的合适载体是本领域技术人员熟知的(参见,例如,Gennaro (编), Remington's Pharmaceutical Sciences(Mack Publishing Company, 1995年第19版))。制剂还可以包括一或多种赋形剂、防腐剂、增溶剂、缓冲剂、防止蛋白丢失到瓶表面上的白蛋白等。
将有效量的包含本发明的二聚蛋白的药物组合物施用受试者。所述二聚蛋白可以通过多种施用模式施用受试者,包括例如通过肌肉内、皮下、静脉内、心房内、关节内、肠胃外、鼻内、肺内、透皮、胸膜内、鞘内和经口的施用途径。对于预防和治疗目的而言,可以如下给受试者施用所述二聚蛋白:单次大剂量递送,在长时间段内连续递送(例如连续透皮递送),或者按照反复施用的方案(例如每小时、每天或每周)。
在该背景下有效剂量的确定,一般是在动物模型研究和之后的人临床试验的基础上,并通过确定在模型受试者中明显减少受试者疾病或障碍的发生率或严重性的有效剂量和施用方案来指导。本发明的组合物的有效剂量随多种不同因素而变动,所述因素包括:施用方式、目标部位、患者生理状态、患者是人还是动物、施用的其它药物、治疗是预防性的还是治疗性的、以及组合物自身的比活和它在个体中引发所需反应的能力。通常,患者是人,但在一些疾病中,患者可以是非人的哺乳动物。一般来说,调节剂量方案以提供最佳的治疗反应,即优化安全性和疗效。相应地,治疗或预防有效量还是这样的量:其中有益效果超过任何不希望的副效果(例如,就二聚的VSTM3蛋白而言,当用二聚的VSTM3蛋白抑制T-细胞介导的免疫应答的有益效果超过任何不希望的副效果时)。就本发明的二聚蛋白(例如,二聚的VSTM3蛋白)的施用而言,剂量范围通常是约0.1 μg-100 mg/kg,或1 μg/kg至约50 mg/kg,更常见地10 μg-5 mg/kg受试者体重。在更具体的实施方案中,药剂的有效量在约1 μg/kg和约20mg/kg之间、约10 μg/kg和约10mg/kg之间、或者约0.1mg/kg和约5mg/kg之间。通过单次或多次施用,包括例如每天多次施用、或每天一次、每周一次、每两周一次或每月一次施用,可以达到在该范围内的剂量。例如,在某些变化中,方案可以包括:开始施用,然后以每周或每两周间隔进行多次后续施用。另一种方案包括:开始施用,然后以每月或每两个月间隔进行多次后续施用。可替换地,按照监测疾病或障碍的临床症状和/或监测疾病生物标志物或其它疾病关联(例如,就T-细胞介导的免疫障碍而言,T细胞活性)的指示,可以不规律地施用。
主治临床医师可以改变药物组合物的剂量,以维持在目标部位处的所需浓度。例如,如果选择静脉内递送模式,根据受试者的状态和预期的测量到的反应,药剂在目标组织处的血流中的局部浓度可以在每升约1-50纳摩尔组合物之间,有时在每升约1.0纳摩尔和每升10、15或25纳摩尔之间。根据递送模式,例如经表皮递送相比于递送至粘膜表面,可以选择更高或更低的浓度。还应当基于所施用的制剂的释放速率来调节剂量,所述制剂例如喷鼻剂相比于粉末、持续释放口服或注射颗粒、透皮制剂等。为了达到相同的血清浓度水平,例如释放速率为5纳摩尔(在标准条件下)的缓释颗粒的施用剂量应当是释放速率为10纳摩尔的颗粒的剂量的约两倍。
可以以液体剂型、气溶胶或固体剂型提供包含本文所述的二聚蛋白(例如,二聚的VSTM3蛋白)的药物组合物。通过可注射的溶液、气溶胶、微滴、局部解剖学(topological)溶液和口服悬浮剂,举例说明液体剂型。示例性固体剂型包括胶囊、片剂和控释剂型。通过微型渗透泵和植入体举例说明后一种剂型(参见,例如,Bremer等人, Pharm. Biotechnol. 10:239, 1997; Ranade, “Implants in Drug Delivery,”Drug Delivery Systems 95-123 (Ranade和Hollinger编, CRC Press 1995);Bremer等人, “Protein Delivery withInfusion Pumps,”Protein Delivery: Physical Systems 239-254 (Sanders和Hendren编, Plenum Press 1997);Yewey等人, “Delivery of Proteins from a ControlledRelease Injectable Implant,”Protein Delivery: Physical Systems 93-117(Sanders和Hendren编, Plenum Press 1997))。其它固体剂型包含乳膏剂、糊剂、其它局部解剖学应用等。
脂质体提供了将治疗性多肽递送至受试者的一种方式,例如,静脉内地、腹膜内地、鞘内地、肌肉内地、皮下地或通过口服施用、吸入或鼻内施用。脂质体是微小囊泡,其由包围水性区室的一层或多层脂质双层构成(一般参见,Bakker-Woudenberg等人, Eur. J. Clin. Microbiol. Infect. Dis. 12 (增刊1):S61, 1993; Kim, Drugs 46:618, 1993;Ranade, “Site-Specific Drug Delivery Using Liposomes as Carriers,”Drug Delivery Systems 3-24 (Ranade和Hollinger编, CRC Press 1995))。脂质体在组成上类似于细胞膜,并且因此,脂质体可安全地施用,且是可生物降解的。依赖于制备的方法,脂质体可以是单层或多层的,脂质体可在直径为0.02 μm至大于10 μm的大小范围内变化。可将各种药剂封装到脂质体中:疏水性试剂被分配到双层中,而亲水性试剂被分配到内部水性空间中(参见,例如,Machy等人,Liposomes In Cell Biology And Pharmacology (JohnLibbey 1987);Ostro等人, American J. Hosp. Pharm. 46:1576, 1989)。此外,通过改变脂质体大小、双层的数目、脂质组成以及脂质体的电荷和表面特征,可以控制被封装的药剂的治疗利用度。
脂质体可吸附至几乎任何类型的细胞上,并随后缓慢地释放被封装的药剂。可替换地,被吸收的脂质体可被吞噬性的细胞内吞。胞吞后,发生脂质体脂质的溶酶体内降解,并释放被封装的药剂(参见Scherphof等人, Ann. N.Y. Acad. Sci. 446:368, 1985)。在静脉内施用后,小脂质体(0.1-1.0 μm)通常被主要位于肝脏和脾脏中的网状内皮系统的细胞吸收,而大于3.0 μm的脂质体则沉积在肺中。网状内皮系统的细胞对较小脂质体的这种优先吸收,已用于将化学治疗剂递送至巨噬细胞和肝脏的肿瘤。
通过几种方法,可以避开网状内皮系统,所述方法包括:使用大剂量的脂质体颗粒饱和,或通过药理学方法选择性地使巨噬细胞失活(参见Claassen等人, Biochim. Biophys. Acta 802:428, 1984)。此外,已经证实,将糖脂衍生的或聚乙二醇衍生的磷脂掺入脂质体膜中,会导致网状内皮系统的吸收显著减少(参见Allen等人, Biochim. Biophys. Acta 1068:133, 1991; Allen等人, Biochim. Biophys. Acta 1150:9,1993)。
通过改变磷脂组成或通过将受体或反受体插入脂质体中,也可制备靶向特定细胞或器官的脂质体。例如,用高含量的非离子表面活性剂制备的脂质体已用于靶向肝脏(参见,例如,Hayakawa等人的日本专利04-244,018 ; Kato等人, Biol. Pharm. Bull. 16:960, 1993)。如下制备这些制剂:在甲醇中混合大豆磷脂酰胆碱、α-生育酚和乙氧基化的氢化蓖麻油(HCO-60),在真空下浓缩该混合物,并随后用水重构该混合物。还已经证实,二棕榈酰磷脂酰胆碱(DPPC)和大豆衍生的甾基葡萄糖苷混合物(SG)和胆固醇(Ch)的脂质体制剂会靶向肝脏(参见Shimizu等人, Biol. Pharm. Bull. 20:881, 1997)。
可替换地,可将各种靶向性反受体结合在脂质体的表面,所述反受体例如抗体、抗体片段、糖、维生素和运输蛋白。例如,就靶向肝脏而言,可用分枝型半乳糖基脂质衍生物修饰脂质体,以使其靶向无唾液酸糖蛋白(半乳糖)受体,该受体只在肝细胞的表面上表达(参见Kato和Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287, 1997; Murahashi等人, Biol. Pharm. Bull.20:259, 1997)。在组织寻靶的更一般方案中,用生物素化的抗体预标记该靶细胞,所述抗体对由靶细胞表达的反受体是特异性的(参见Harasym等人, Adv. Drug Deliv. Rev. 32:99, 1998)。在游离抗体的血浆清除之后,施用链霉亲和素缀合的脂质体。在另一个方案中,将靶向性抗体直接附着于脂质体上(参见Harasym等人, 出处同上)。
使用蛋白质微囊化的标准技术,可以将多肽封装在脂质体中(参见,例如,Anderson等人, Infect. Immun. 31:1099, 1981; Anderson等人, Cancer Res. 50:1853, 1990; Cohen等人, Biochim. Biophys. Acta 1063:95, 1991; Alving等人,“Preparation and Use of Liposomes in Immunological Studies,”Liposome Technology (第III卷) 317 (Gregoriadis编, CRC Press,1993年第2版);Wassef等人, Meth. Enzymol. 149:124, 1987)。如上面所指出的,治疗上有用的脂质体可包含各种组分。例如,脂质体可包含聚(乙二醇)的脂质衍生物(参见Allen等人, Biochim. Biophys. Acta 1150:9, 1993)。
已经设计了可降解的聚合物微球,以维持治疗性蛋白的高全身水平。用可降解的聚合物(诸如聚(丙交酯-共聚-乙交酯) (PLG)、聚酸酐、聚(原酸酯))、不可生物降解的乙基乙酸乙烯酯聚合物制备微球,其中蛋白被捕获在聚合物中(参见,例如,Gombotz andPettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role of Polymers in DrugDelivery,”Drug Delivery Systems 51-93 (Ranade和Hollinger编, CRC Press 1995);Roskos和Maskiewicz, “Degradable Controlled Release Systems Useful for ProteinDelivery,”Protein Delivery: Physical Systems 45-92 (Sanders和Hendren编,Plenum Press 1997);Bartus等人, Science 281:1161, 1998; Putney和Burke, Nature Biotechnology 16:153, 1998; Putney, Curr. Opin. Chem. Biol. 2:548, 1998)。聚乙二醇(PEG)包被的纳米球还可以提供用于治疗性蛋白的静脉内施用的载体(参见,例如,Gref等人, Pharm. Biotechnol. 10:167, 1997)。
本领域技术人员可设计其它剂型,例如由例如,Ansel和Popovich,Pharmaceutical Dosage Forms and Drug Delivery Systems (Lea和Febiger, 1990年第5版);Gennaro (编), Remington's Pharmaceutical Sciences (Mack PublishingCompany, 1995年第19版), 以及Ranade和Hollinger, Drug Delivery Systems (CRCPress 1996)所显示的。
如本文所述的二聚蛋白,包括例如二聚的VSTM3 (B7R1) 蛋白,可以用于基因治疗的背景下。基因治疗可以广义地定义为:将遗传物质转移进细胞中,以暂时地或永久地改变所述细胞的表型。正在开发众多的方法,用于通过基因治疗将细胞因子、肿瘤抗原和其它共刺激性分子递送至肿瘤患者内的特定位置(一般参见Rosenberg (编), Principles and practice of the biologic therapy of cancer (Lippincott Williams & Wilkins,Philadelphia, PA, 2000年第3版))。可以改进这些方法,以使用编码本发明的多肽融合体的DNA或RNA。
因此,在有些实施方案中,通过施用编码如本文所述的多肽融合体的核酸,治疗受试者中的疾病或障碍。例如,在某些实施方案中,通过施用编码如本文所述的VSTM3 (B7R1)多肽融合体的核酸,抑制受试者中的T-细胞介导的应答;使用这样的编码VSTM3的核酸,可以象上面一般地讨论的那样,治疗T-细胞介导的免疫障碍。就核酸治疗而言,表达如本文所述的多肽融合体,并从细胞分泌,以形成二聚蛋白,所述二聚蛋白发挥治疗效果的方式类似于如上所述直接施用给受试者的本发明的二聚蛋白(例如,就编码VSTM3的核酸而言,表达如本文所述的VSTM3多肽融合体,并分泌,以形成二聚的VSTM3蛋白,后者抑制T-细胞介导的效应的方式类似于直接施用给受试者的二聚的VSTM3蛋白)。可替换地,可以以特定形式表达本发明的多肽融合体,所述形式维持与表达所述蛋白的细胞的表面(例如,与功能性跨膜结构域或GPI连接)的结合;就促进靶向特定细胞或组织以维持局部化的治疗效果(例如,通过二聚的VSTM3蛋白的表达,局部化地抑制T-细胞介导的应答)而言,这样的实施方案是特别有用的。
用于治疗方法中的编码多肽的核酸可以是DNA或RNA。编码如本文所述的多肽融合体的核酸区段通常连接至调控元件(诸如启动子和增强子),后者允许在患者的预期靶细胞中表达所述DNA区段。例如,就在血细胞中表达(通过VSTM3多肽的表达来抑制T-细胞介导的应答所希望的)而言,得自轻链或重链免疫球蛋白基因的启动子和增强子元件或CMV主要中间早期启动子和增强子适用于指导表达。经常将连接的调控元件和编码序列克隆进载体中。
可得到许多病毒载体系统,包括逆转录病毒系统(参见,例如,Lawrie和Tumin,Cur. Opin. Genet. Develop. 3, 102-109, 1993);腺病毒载体(参见,例如,Bett等人,J. Virol. 67, 5911, 1993);腺相关病毒载体(参见,例如,Zhou等人, J. Exp. Med.179, 1867, 1994),得自包括痘苗病毒和禽痘病毒在内的痘家族的病毒载体,得自α病毒属的病毒载体,诸如源自Sindbis和Semliki森林病毒的那些(参见,例如,Dubensky等人, J.Virol. 70, 508-519, 1996)和乳头状瘤病毒(Ohe等人, Human Gene Therapy 6, 325-333, 1995; WO 94/12629 (Woo等人);Xiao & Brandsma, Nucleic Acids. Res. 24,2630-2622, 1996)。
可以将编码本发明的多肽融合体的DNA或含有所述DNA的载体包装进脂质体中。US5,208,036、5,264,618、5,279,833和5,283,185描述了合适的脂质和有关的类似物。还可以用微粒载体吸附或结合编码所述多肽融合体的载体和DNA,所述微粒载体的实例包括聚甲基丙烯酸甲酯聚合物和聚丙交酯和聚(丙交酯-共聚-乙交酯) (参见,例如,McGee等人,J. Micro Encap., 1996)。
通过施用给单个患者,通常通过全身性施用(例如,静脉内的、腹膜内的、鼻的、胃的、真皮内的、肌肉内的、真皮下的或颅内的输注)或局部施用(参见例如,US 5,399,346),可以在体内递送基因治疗载体或裸DNA。使用基因枪,也可以施用DNA。(参见Xiao &Brandsma, 出处同上)。将编码多肽的DNA沉淀到微型金属珠粒的表面上。用冲击波或扩张的氦气加速微射弹,并穿透组织至几个细胞层的深度。例如,由Agacetus, Inc. MiddletonWI生产的AccelTM基因递送装置是合适的。可替换地,简单地通过将DNA点在受化学或机械刺激的皮肤上,裸DNA可以穿过皮肤进入血流中(参见,例如,WO 95/05853)。
如本文所述的药物组合物也可以用在联合治疗的背景下。术语“联合治疗”在本文中用于表示,给受试者施用至少一种治疗有效剂量的本文所述的二聚蛋白和其它治疗剂。
药物组合物可以作为试剂盒供应,所述试剂盒包括容器,所述容器包含如本文所述的多肽融合体、二聚蛋白或多核苷酸。可以提供治疗性分子,例如,以用于单剂或多剂的注射液的形式,或作为将在注射前重构的无菌粉末。可替换地,这样的试剂盒可以包括用于施用治疗性蛋白或多核苷酸的干粉分配器、液体气溶胶发生器或喷雾器。这样的试剂盒可另外包括关于药物组合物的适应症和用法的书面信息。例如,在包含VSTM3 (B7R1) 组合物的试剂盒的具体实施方案中,这样的信息可以包括下述声明:在已知对VSTM3有超敏反应的患者中,禁用所述VSTM3组合物。
下述非限制性实施例进一步例证了本发明。
实施例1
鼠B7R1 Barbell (mB7R1-Barbell)的构建和表达
使用两步法,通过同源重组,构建了含有mB7R1-mFc2-mB7R1(具有天然mB7R1前导序列的mB7R1-Barbell;多核苷酸序列显示在SEQ ID NO:9中;编码的多肽序列显示在SEQID NO:10中)的表达质粒。
步骤1
首先,使用以前制备的克隆作为模板,通过PCR扩增,制备含有融合蛋白mB7R1-mFc2 (与小鼠Fc片段(mFc2)融合的小鼠B7R1细胞外结构域(ECD)) 的序列的DNA片段。所述mB7R1-mFc2片段具有在pZMP42中的序列重叠,并使用引物zc60639 (SEQ ID NO:36)、zc60643 (SEQ ID NO:37)和zc60645 (SEQ ID NO:38)制备。使用下述PCR条件,制备该片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利申请公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。将100微升感受态酵母(酿酒酵母) 细胞分别与10µl插入物DNA和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories, Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100-µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 mlH2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002) 的P1 (得自QIAPREP®Spin Miniprep Kit,Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。
所述构建体用作构建的第二步的基础。
步骤2
构建的第二步涉及:使用以前制备的克隆作为模板,制备mB7R1 ECD片段。所述mB7R1 ECD片段具有添加在所述片段的5’末端上的BspEI限制切割位点和添加在所述片段的3’末端上的Bsu36I位点,并使用引物zc60642 (SEQ ID NO:39)和zc60641 (SEQ ID NO:40)制备。使用下述PCR条件,制备所述片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用BspEI和Bsu36I,消化得自所述第一步的构建体和所述第二步mB7R1 ECD片段。使所述第一步构建体和所述第二步mB7R1 ECD片段的消化过的DNA在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与所述DNA的大小相对应的带进行凝胶提取。然后,使用本领域已知的方法,将两种纯化的DNA制备物连接到一起。
使用1µl连接制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC (2%BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl, 2.5mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco), 100mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。该克隆命名为#1863。
mB7R1-Barbell(具有天然信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:9和10中。mB7R1-Barbell的成熟形式与SEQ ID NO:10的氨基酸26-489 (由SEQ ID NO:9的核苷酸76-1467编码)相对应。
mB7R1-Barbell的表达
3组200µg构建体#1863各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6%CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例2
人B7R1 Barbell (B7R1-Barbell)的构建和表达
使用两步法,通过同源重组,构建了含有B7R1-Fc5-B7R1 (具有天然B7R1前导序列的B7R1-Barbell;多核苷酸序列显示在SEQ ID NO:5中;编码的多肽序列显示在SEQ ID NO:6中) 的表达质粒。
步骤1
首先,通过PCR扩增和重组,制备了含有融合蛋白B7R1-Fc5 (与不具有效应物功能的Fc片段Fc5融合的人B7R1细胞外结构域(ECD))的序列的DNA片段。使用以前制备的克隆作为模板,制备B7R1 ECD片段和Fc5片段。B7R1 ECD片段具有在Fc5中的序列重叠,并使用引物zc53051 (SEQ ID NO:41)和zc60385 (SEQ ID NO:42)制备。编码Fc5的片段具有:在B7R1ECD中的5’重叠、GGGSG接头、含有BspEI位点和下游BglII位点的多克隆区、和与pZMP42载体序列的序列重叠,并使用引物zc60386 (SEQ ID NO:43)、zc59433 (SEQ ID NO:44)和zc59434 (SEQ ID NO:45)制备。使用下述PCR条件,制备这2个片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利申请公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。将100微升感受态酵母(酿酒酵母) 细胞分别与10µl插入物DNA和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories, Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100-µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 mlH2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002)的P1 (得自QIAPREP®Spin Miniprep Kit,Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。
将该构建体命名为#1795,并建立用作构建的第二步的基础。
步骤2
构建了构建体#1795,作为在pZMP42中建立人B7R1 Barbell载体的第一步。#1795含有在载体pZMP42中的B7R1-Fc5片段,所述载体pZMP42包括:由接头序列组成的3’添加段,和含有限制位点BspEI和BglII的多克隆区。
构建的第二步涉及:使用以前制备的克隆作为模板,制备B7R1 ECD片段。所述B7R1ECD片段具有:添加在所述片段的5’末端上的BspEI限制切割位点和添加在所述片段的3’末端上的BglII位点,并使用引物zc59435 (SEQ ID NO:77)、zc60392 (SEQ ID NO:78)和zc59434 (SEQ ID NO:45)制备。使用下述PCR条件,制备所述片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用BspEI和BglII,消化构建体#1795和第二步B7R1 ECD片段。使构建体#1795和第二步B7R1 ECD片段的消化的DNA在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与所述DNA的大小相对应的带进行凝胶提取。然后,使用本领域已知的方法,将两种纯化的DNA制备物连接到一起。
使用1µl连接制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC (2%BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl, 2.5mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco), 100mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。该克隆命名为#1812。
B7R1-Barbell(具有天然信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:5和6中。B7R1-Barbell的成熟形式与SEQ ID NO:6的氨基酸22-498相对应(由SEQ ID NO:5的核苷酸64-1494编码)。
B7R1-Barbell的表达
3组200µg构建体#1812各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6%CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例3
鼠B7R1 Tandem (mB7R1-Tandem)的构建和表达
通过连接进以前改进形式的含有mFc2插入片段的表达载体pZMP42(在实施例1的步骤1中描述的构建体)中,构建了含有mB7R1m-mB7R1-mFc2 (具有天然mB7R1前导序列的mB7R1-Tandem;多核苷酸序列显示在SEQ ID NO:11中;编码的多肽序列显示在SEQ ID NO:12中) 的表达质粒。
使用以前制备的克隆作为模板,制备了含有mB7R1细胞外结构域(ECD) 的DNA片段。所述片段具有:工程改造进它的5’末端的EcoRI位点、mB7R1 ECD和Gly-Ser接头,并使用引物zc60639 (SEQ ID NO:36)、zc60640 (SEQ ID NO:46)和zc60644 (SEQ ID NO:47)制备。制备第二种DNA片段,其具有:在3’末端上与所述第一种片段的Gly-Ser接头的重叠、mB7R1 ECD和在该片段的3’末端上的Bgl II位点,并使用引物zc60642 (SEQ ID NO:48)和zc28844 (SEQ ID NO:49)制备。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICK™凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用重叠PCR,使这2个DNA片段融合成单个片段。将所述2个凝胶纯化的片段加入PCR试管中,并使用引物zc60639 (SEQ ID NO:36)和zc28844 (SEQ ID NO:49)扩增。得到的片段含有侧接核心序列mB7R1-mB7R1的5’EcoRI位点和3’Bgl II位点。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用EcorI和BglII,消化纯化的片段和改进的载体pZMP42,分离感兴趣的带,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704)进行凝胶纯化。然后使用本领域已知的方法,连接纯化的片段,使得mB7R1-mB7R1片段与在改进的pZMP42载体中含有的mFc2同框。得到的构建体可以表达由mB7R1-mB7R1-mFc2组成的蛋白。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (专利公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
使用1µl连接DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC (2%BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl, 2.5mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco), 100mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。最终的克隆命名为构建体#1864。
mB7R1-Tandem(具有天然信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:11和12中。mB7R1-Tandem的成熟形式与SEQ ID NO:12的氨基酸26-504或26-503(分别由SEQ ID NO:11的核苷酸76-1512或76-1509编码)相对应。
mB7R1-Tandem的表达
3组200µg构建体#1864各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6%CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例4
人B7R1 Tandem (B7R1-Tandem)的构建和表达
通过连接进以前制备的构建体中(所述构建体被命名为构建体#1795 (在实施例2的步骤1中描述的构建体),其含有在表达载体pZMP42中的B7R1-Fc5插入片段)构建含有B7R1-B7R1-Fc5 (具有天然B7R1前导序列的B7R1-Tandem;多核苷酸序列显示在SEQ ID NO:7中;编码的多肽序列显示在SEQ ID NO:8中) 的表达质粒。以两步法进行构建。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (专利公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
步骤1
使用以前制备的克隆作为模板,制备含有B7R1细胞外结构域(ECD)的DNA片段。所述片段具有:工程改造进它的5’末端中的EcoRI位点、B7R1 ECD、引入BspEI位点中的Gly-Ser接头,并使用引物zc53051 (SEQ ID NO:41)、zc62529 (SEQ ID NO:50)和zc62530 (SEQID NO:51) 制备。使用下述PCR条件,制备该片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用EcoRI和BspEI,消化构建体#1795和纯化的PCR片段。使构建体#1795和所述PCR片段的消化的DNA在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen,目录号28704),对与所述DNA的大小相对应的带进行凝胶提取。然后,使用本领域已知的方法,将两种纯化的DNA制备物连接到一起。
使用1µl连接DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC (2%BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl, 2.5mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco), 100mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。所述构建体用作构建的第二步的基础。
步骤2
构建的第二步涉及:使用以前制备的克隆作为模板,制备B7R1-Fc5片段(与不具有效应物功能的Fc片段Fc5融合的人B7R1细胞外结构域(ECD))。所述B7R1-Fc5片段具有添加在所述片段的5’末端上的BspEI限制切割位点和添加在所述片段的3’末端上的Bgl II位点,并使用引物zc62531 (SEQ ID NO:52)和zc62532 (SEQ ID NO:53) 制备。使用下述PCR条件,制备所述片段:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
使用BspEI和Bgl II,消化得自所述第一步的构建体和所述第二步B7R1-Fc5片段。使所述第一步构建体和所述第二步B7R1-Fc5片段的消化的DNA在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与所述DNA的大小相对应的带进行凝胶提取。然后,使用本领域已知的方法,将两种纯化的DNA制备物连接到一起。
使用1µl连接制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC (2%BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl, 2.5mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco), 100mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。所述克隆命名为#1914。
B7R1-Tandem(具有天然信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:7和8中。B7R1-Tandem的成熟形式与SEQ ID NO:8的氨基酸22-513或22-512 (分别由SEQ ID NO:7的核苷酸64-1539或64-1536编码)相对应。
B7R1-Tandem的表达
3组200µg构建体#1914各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6%CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例5
使用otPA前导序列的含有G25成熟起点的人B7R1 Barbell(B7R1[G25-P141]-Barbell)的构建和表达
使用3个DNA片段(它们当组合时含有B7R1[G25-P141]-Barbell的序列)和表达载体pZMP42,通过同源重组,构建了含有B7R1[G25-P141]-Fc5-B7R1[G25-P141] (B7R1[G25-P141]-Barbell;多核苷酸序列显示在SEQ ID NO:13的残基106-1518中;编码的多肽序列显示在SEQ ID NO:14的残基36-506中) 的表达质粒。
使用以前制备的B7R1-Barbell的克隆作为模板,通过3个片段的PCR扩增,制备B7R1[G25-P141]-Barbell片段。使用寡核苷酸zc64230 (SEQ ID NO:54)和zc64219 (SEQID NO:55),制备第一种片段,其包括:所述载体的5’侧接序列,直至残基C69的第一种B7R1细胞外结构域(ECD)模块的部分,和与C69以后的残基的重叠序列。使用寡核苷酸zc64215(SEQ ID NO:56)和zc64216 (SEQ ID NO:57),制备第二种片段,其范围为:从在所述第一种片段中的侧接序列,穿过第一种B7R1模块的残基C69,到Fc5的末端。使用寡核苷酸zc64228(SEQ ID NO:58)、zc64220 (SEQ ID NO:59)、zc64224 (SEQ ID NO:60)、zc64231 (SEQ IDNO:61)、zc64225 (SEQ ID NO:62)、zc64221 (SEQ ID NO:63)、zc64226 (SEQ ID NO:64)、zc64222 (SEQ ID NO:65)、zc64223 (SEQ ID NO:66)、zc64258 (SEQ IN NO:67)、和zc59434 (SEQ ID NO:45),通过重叠PCR,合成地制备最终片段。当通过酵母重组进行装配时,所述3个片段的融合体包括:与pZMP42载体的5’重叠区,otPA前导序列, 第一种B7R1[G25-P141]模块(与Fc5融合,与第二种B7R1[G25-P141]模块融合),和与pZMP42载体序列的3’重叠区。用于制备每个片段的PCR条件如下:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利申请公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。使100微升感受态酵母(酿酒酵母) 细胞各自与3µl每种插入DNA片段和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories, Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 mlH2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002)的P1 (得自QIAPREP®Spin Miniprep Kit,Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。将所述构建体命名为构建体#2024。
B7R1[G25-P141]-Barbell(具有otPA信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:13和14中。B7R1[G25-P141]-Barbell的成熟形式与SEQID NO:14的氨基酸36-506 (由SEQ ID NO:13的核苷酸106-1518编码)相对应。
B7R1[G25-P141]-Barbell的表达
3组200µg构建体各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6% CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例6
使用otPA前导序列的含有G25成熟起点和C69Y突变的人B7R1 Barbell(B7R1[G25-P141][C69Y]-Barbell)的构建和表达
使用3个DNA片段(它们当组合时含有B7R1[G25-P141][C69Y]-Barbell的序列)和表达载体pZMP42,通过同源重组,构建了含有B7R1[G25-P141][C69Y]-Fc5-B7R1[G25-P141][C69Y](B7R1[G25-P141][C69Y]-Barbell;多核苷酸序列显示在SEQ ID NO:13的残基106-1518中;编码的多肽序列显示在SEQ ID NO:16的残基36-506中)的表达质粒。
使用以前制备的B7R1-Barbell克隆作为模板,通过3个片段的PCR扩增,制备B7R1[G25-P141][C69Y]-Barbell片段。使用寡核苷酸zc64230 (SEQ ID NO:54)和zc64218 (SEQID NO:68),制备第一种片段,其包括:所述载体的5’侧接序列,直至残基C69的第一种B7R1细胞外结构域(ECD)模块的部分,和与C69以后的残基的重叠序列。使用寡核苷酸zc64215(SEQ ID NO:56)和zc64216 (SEQ ID NO:57),制备第二种片段,其范围为:从在所述第一种片段中的侧接序列,穿过第一种B7R1模块的残基C69,到Fc5的末端。使用寡核苷酸zc64228(SEQ ID NO:58)、zc64220 (SEQ ID NO:59)、zc64224 (SEQ ID NO:60)、zc64227 (SEQ IDNO:69)、zc64225 (SEQ ID NO:62)、zc64221 (SEQ ID NO:63)、zc64226 (SEQ ID NO:64)、zc64222 (SEQ ID NO:65)、zc64223 (SEQ ID NO:66)、zc64258 (SEQ ID NO:67)和zc59434(SEQ ID NO:45),通过重叠PCR,合成地制备最终片段。当通过酵母重组进行装配时,所述3个片段的融合体包括:与pZMP42载体的5’重叠区,otPA前导序列, 第一种B7R1[G25-P141][C69Y]模块(与Fc5融合,与第二种B7R1[G25-P141][C69Y]模块融合),和与pZMP42载体序列的3’重叠区。用于制备每个片段的PCR条件如下:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
所述插入片段含有B7R1 ECD模块的改进形式。残基C69被突变为酪氨酸(Y)。成熟蛋白的N-端起点也从人预测起点(氨基酸残基22)调至氨基酸残基25 (G25)。进行这些变化,以克服生产人蛋白时观察到的几个问题。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。使100微升感受态酵母(酿酒酵母) 细胞各自与3µl每种插入DNA片段和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories, Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 mlH2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002)的P1 (得自QIAPREP®Spin Miniprep Kit,Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。将所述构建体命名为构建体#2026。
B7R1[G25-P141][C69Y]-Barbell(具有otPA信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:15和16中。B7R1[G25-P141]-Barbell的成熟形式与SEQ ID NO:16的氨基酸36-506(由SEQ ID NO:15的核苷酸106-1518编码)相对应。
B7R1[G25-P141][C69Y]-Barbell的表达
3组200µg构建体各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6% CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例7
使用EMIL前导序列的含有G25成熟起点和C69Y突变的人B7R1 Barbell(B7R1[G25-P141][C69Y]-Barbell)的构建和表达
使用含有B7R1[G25-P141][C69Y]-Barbell的序列的DNA片段和表达载体pZMP42,通过同源重组,构建了含有B7R1[G25-P141][C69Y]-Fc5-B7R1[G25-P141][C69Y](B7R1[G25-P141][C69Y]-Barbell;多核苷酸序列显示在SEQ ID NO:17的残基67-1497中;编码的多肽序列显示在SEQ ID NO:18的残基23-493中)的表达质粒。使用引物zc65030 (SEQ IDNO:70)、zc65029 (SEQ ID NO:71)和zc59434 (SEQ ID NO:45),通过PCR扩增,制备B7R1[G25-P141][C69Y]-Barbell片段。
使用以前制备的B7R1[G25-P141][C69Y]-Barbell的克隆作为模板,制备B7R1[G25-P141][C69Y]-Barbell片段,命名为构建体#2026。所述片段包括:与pZMP42载体序列的5’重叠区,命名为“EMIL”的前导序列(SEQ ID NO:17的残基1-66;编码的氨基酸序列显示在SEQ ID NO:18的残基1-22中),B7R1[G25-P141][C69Y]-Barbell区段(SEQ ID NO:17的残基67-1497;编码的氨基酸序列显示在SEQ ID NO:18的残基23-493中),和与pZMP42载体序列的3’重叠区。使用的PCR条件如下:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
所述插入片段含有B7R1细胞外结构域(ECD) 模块的改进形式。残基C69被突变为酪氨酸(Y)。成熟蛋白的N-端起点也从人预测起点(氨基酸残基22)调至氨基酸残基25(G25)。进行这些变化,以克服生产人蛋白时观察到的几个问题。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利申请公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。将100微升感受态酵母(酿酒酵母) 细胞分别与10µl插入物DNA和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories, Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 mlH2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002)的P1 (得自QIAREP®Spin Miniprep Kit,Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。将所述构建体命名为构建体#2065。
B7R1[G25-P141][C69Y]-Barbell(具有EMIL信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:17和18中。B7R1[G25-P141][C69Y]-Barbell的成熟形式与SEQ ID NO:18的氨基酸23-493 (由SEQ ID NO:17的核苷酸67-1497编码)相对应。
B7R1[G25-P141][C69Y]-Barbell的表达
3组200µg构建体各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6% CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例8
使用EMIL前导序列的含有G25成熟起点和C69Y突变的人B7R1 Tandem(B7R1[G25-P141][C69Y]-Tandem)的构建和表达
使用含有B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y]的序列的DNA片段(SEQID NO:20的残基23-139)、编码Fc5的DNA片段和表达载体pZMP42,通过同源重组,构建了含有B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y]-Fc5 (B7R1[G25-P141][C69Y]-Tandem;多核苷酸序列显示在SEQ ID NO:19的残基67-1524中;编码的多肽序列显示在SEQ ID NO:20的残基23-508中) 的表达质粒。使用寡核苷酸zc65030 (SEQ ID NO:70)、zc65029 (SEQID NO:71)、zc65050 (SEQ ID NO:72)、zc65051 (SEQ ID NO:73)和zc65052 (SEQ ID NO:74),通过一系列PCR扩增,制备B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y] 片段。使用寡核苷酸zc65053 (SEQ ID NO:75)和zc65054 (SEQ ID NO:76),制备Fc5片段。
使用以前制备的B7R1[G25-P141][C69Y]-Fc5-B7R1[G25-P141][C69Y](B7R1[G25-P141][C69Y]-Barbell)的克隆作为模板,制备B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y] 片段,命名为构建体#2026 (参见实施例6)。第一个反应使用寡核苷酸zc65030(SEQ ID NO:70)、zc65029 (SEQ ID NO:71)和zc65050 (SEQ ID NO:72),扩增一个具有5’EMIL前导序列的B7R1[G25-P141][C69Y]单元。第二个反应使用寡核苷酸zc65051 (SEQ IDNO:73)和zc65052 (SEQ ID NO:74),扩增第二个B7R1[G25-P141][C69Y]单元。最后一个反应使用寡核苷酸zc65030和zc65052对前两个扩增产物进行重叠PCR,以建立单个融合的B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y] 片段。所述片段包括:与pZMP42载体序列的5’重叠区,命名为“EMIL”的前导序列(SEQ ID NO:19的残基1-66;编码的氨基酸序列显示在SEQ ID NO:20的残基1-22中),通过Gly-Ser接头连接的B7R1[G25-P141][C69Y]的2个连续拷贝(SEQ ID NO:19的残基67-828;编码的氨基酸序列显示在SEQ ID NO:20的残基23-276中),和与Fc5的5’末端的3’重叠区。使用的所有反应的PCR条件如下:1个在94℃保持5分钟的循环;35个在94℃保持1分钟、然后在58℃保持2分钟、然后在72℃保持3分钟的循环;1个在72℃保持10分钟的循环。
使用引物zc65053 (SEQ ID NO:75)和zc65054 (SEQ ID NO:76),制备编码Fc5的第二种片段(SEQ ID NO:19的残基829-1524;编码的氨基酸序列显示在SEQ ID NO:20的残基277-508中)。该片段包括Fc5片段和与载体pZMP42的3’重叠区。
使所述PCR反应混合物在1% 琼脂糖凝胶上泳动,并使用QIAQUICKTM凝胶提取试剂盒(Qiagen, 目录号28704),对与插入片段的大小相对应的带进行凝胶提取。
所述插入片段含有B7R1细胞外结构域(ECD) 模块的改进形式。残基C69被突变为酪氨酸(Y)。成熟蛋白的N-端起点也从人预测起点(氨基酸残基22)调至氨基酸残基25(G25)。进行这些变化,以克服生产人蛋白时观察到的几个问题。
质粒pZMP42是哺乳动物表达载体,其含有:表达盒,所述表达盒具有MPSV启动子、用于插入编码序列的多个限制位点和otPA信号肽序列;得自丙型肝炎病毒的内部核糖体进入位点(IRES)元件,和在跨膜结构域的C-端末端截短的CD8的细胞外结构域;得自脊髓灰质炎病毒的内部核糖体进入位点(IRES)元件,DHFR基因,和SV40终止子;大肠杆菌复制起点;和在酿酒酵母中的选择和复制所需的URA3和CEN-ARS序列。它构建自pZMP21 (美国专利申请公开号US 2003/0232414 A1) (保藏在美国典型培养物保藏中心, 10801 UniversityBoulevard, 马纳萨斯, VA 20110-2209,命名为ATCC# PTA-5266)。
在与PCR片段一起在酵母中重组之前,用BglII切割质粒pZMP42。使100微升感受态酵母(酿酒酵母) 细胞各自与5µl B7R1[G25-P141][C69Y]-B7R1[G25-P141][C69Y] 片段DNA、5µl Fc5片段DNA和100 ng切割过的pZMP42载体相混合,并将所述混合物转移至0.2-cm电穿孔容器。使用0.75 kV (5 kV/cm)、∞欧姆和25µF的电源(BioRad Laboratories,Hercules, CA) 设置,对所述酵母/DNA混合物进行电脉冲。将600µl 1.2 M山梨醇加入所述容器中,将所述酵母以100µl和300µl等分试样铺板在2个URA-D平板上,并在30℃孵育。约72小时以后,将得自单个平板的Ura+ 酵母转化物再悬浮于1 ml H2O中,并简单地离心,以沉淀出酵母细胞。通过在0.1 ml裂解缓冲液(2% Triton X-100, 1% SDS, 100 mM NaCl, 10mM Tris, pH 8.0, 1 mM EDTA)和0.1 mL加入10单位的藤黄节杆菌酶(Zymo Research, 目录号E1002)的P1 (得自QIAPREP®Spin Miniprep Kit, Qiagen, 目录号27106)中涡旋,再悬浮所述细胞沉淀物。将所述酵母悬浮液在37℃水浴中孵育10分钟。使用标准的QIAPREP®Spin Miniprep Kit方案(Qiagen, 目录号27106),从加入试剂P2的步骤开始,从酵母分离DNA。
使用5 μl酵母DNA制备物和50 μl细胞,进行电感受态的大肠杆菌宿主细胞(DH12S)的转化。在2.0 kV、25µF和400欧姆,对细胞进行电脉冲。电穿孔以后,加入1 ml SOC(2% BACTOTM胰蛋白胨(Difco, Detroit, MI), 0.5% 酵母提取物(Difco), 10 mM NaCl,2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM葡萄糖),然后,将所述细胞以50µl和200µl等分试样铺板在2个LB AMP平板(LB液体培养基(Lennox), 1.8% BACTOTM琼脂(Difco),100 mg/L氨苄西林)上。
对所述构建体的几个克隆的插入物进行序列分析,并选择一个含有正确序列的克隆。根据生产商的说明书,使用商购可得的试剂盒(QIAGEN Plasmid Mega Kit, Qiagen,巴伦西亚, CA),分离大规模质粒DNA。将所述构建体命名为构建体#2066。
B7R1[G25-P141][C69Y]-Tandem(具有EMIL信号序列)的全长核苷酸编码序列和对应的氨基酸序列分别显示在SEQ ID NO:19和20中。B7R1[G25-P141][C69Y]-Tandem的成熟形式与SEQ ID NO:20的氨基酸23-508或23-507 (分别由SEQ ID NO:19的核苷酸67-1524或67-1521编码)相对应。
B7R1[G25-P141][C69Y]-Tandem的表达
3组200µg构建体各自用200单位的Pvu I在37℃消化3小时,然后,用IPA沉淀,并在1.5 mL微量离心管中离心。从沉淀物中倾析出上清液,用1 mL 70% 乙醇洗涤沉淀物,并在室温孵育5分钟。将所述试管在微量离心机中在14,000 RPM离心10分钟,并从沉淀物中倾析出上清液。然后在无菌环境中将所述沉淀物再悬浮于750µl ZF1介质中,在60℃孵育10分钟,并使其冷却至室温。在3个试管中的每一个中沉淀出5 x 106个CHO DXB11 5xSA细胞,并使用DNA-介质溶液再悬浮。将所述DNA/细胞混合物放入0.4 cm间隙容器中,并使用下述参数进行电穿孔:950µF,高电容,和300 V。然后取出所述容器的内容物,合并,并用ZF1介质稀释至25 mL,放入125 mL摇瓶中。将所述烧瓶放入培养箱中的振荡器上,并在37℃,6% CO2,120 RPM振荡。
对细胞系进行营养物选择,随后逐步放大至200nM甲氨蝶呤(MTX)。通过蛋白质印迹验证表达,并放大所述细胞系,随后进行蛋白纯化。
实施例9
人和小鼠B7R1 Tandem和Barbell蛋白的纯化
以两种构型Tandem和Barbell (参见上述实施例),使人和小鼠可溶性的B7R1细胞外结构域与人Fc融合,用于瞬时和稳定表达。从转染的293或CHO细胞生产人B7R1 Fc融合蛋白,并从转染的CHO细胞生产小鼠蛋白。使用本领域已知的方法进行转染。小鼠蛋白一致地在比人形式更高的水平生成,直到工程改造人形式以实现更高产率。(将人形式工程改造成,具有在天然B7R1细胞外结构域的甘氨酸25 (G25) 处的成熟起点,以及在位置69处引入半胱氨酸至酪氨酸的突变(C69Y);参见,例如,实施例7和8, 出处同上)。使用相同方法纯化所有形式,所述方法包括:亲和捕获,随后使用大小排阻色谱法浓缩,用于缓冲液更换。就人和小鼠B7R1 Fc融合蛋白而言,在1.5-10 L实验室小试规模进行纯化。
表3: 人和小鼠B7R1 Tandem和Barbell的纯化
收获细胞培养上清液,并用0.2µm过滤器无菌过滤。通过蛋白A琼脂糖或MabSelectSURE和Superdex 200大小排阻色谱法(都得自GE Healthcare, Piscataway, NJ)的组合,从过滤的介质中纯化蛋白。根据规模,用3柱体积(CV) 的25 mM柠檬酸钠-磷酸钠、250 mM硫酸铵pH 3缓冲液预洗脱5-157 ml蛋白A柱,并用20 CV 25 mM柠檬酸钠-磷酸钠、250 mM硫酸铵pH 7.2平衡。以24–42cm/小时,将CHO培养物上清液直接上样至蛋白A柱在4℃过夜,以捕获在上清液中的B7R1 Fc融合蛋白。上样结束以后,用至少10 CV的平衡缓冲液洗涤柱。接着,用至少10 CV的25 mM柠檬酸钠-磷酸钠、250 mM硫酸铵pH 7.2缓冲液洗涤柱,此后,在92-149 cm/小时,用20 CV的从pH 7.2至pH 3的梯度(使用柠檬酸盐-磷酸盐-硫酸铵缓冲液形成)洗脱结合的蛋白。将含有靶物的级分收集进装有2.0 M Tris(pH 8.0)的试管中,以便立即中和洗脱的蛋白。基于洗脱曲线上的A280拐折,合并所述级分。
接着通过超滤将亲和合并物(affinity pool)浓缩至小于大小排阻柱体积的3%,所述超滤使用Amicon Ultra-15 30K NWML离心装置(Millipore)或具有30kD MW截止膜的搅拌单元。然后,在28 cm/小时,将浓缩物注射到适当大小的Superdex 200柱上,所述柱在35 mM磷酸钠、120 mM NaCl(pH 7.3)中预平衡。基于A280,合并含有纯化的B7R1 Fc融合蛋白的级分,通过0.2µm过滤器过滤,测定含量和内毒素,并在-80℃冷冻为等分试样。使用理论消光系数,通过在A280的吸光度,确定最终的纯化蛋白的含量。总方法回收率范围是10-14 mg /L(就小鼠蛋白而言)和6-66 mg/L(就人构建体而言)。
纯化的B7R1 Tandem和Barbell蛋白的分析
通过SDS-PAGE (4-12% BisTris, Invitrogen, Carlsbad, CA)(用0.1% 考马斯R250进行蛋白染色),和通过使用抗-IgG-HRP的免疫印迹法,表征重组B7R1 Fc融合蛋白。使用Invitrogen Novex的Xcell II mini-cell,对纯化的蛋白进行电泳,并在含有25 mMTris碱、200 mM甘氨酸和20% 甲醇的缓冲液中在环境温度在600 mA保持45分钟而转移至硝酸纤维素(0.2 mm; Invitrogen, Carlsbad, CA)。然后用在50 mM Tris、150 mM NaCl、5mM EDTA、0.05% Igepal (TBS)中的10% 脱脂奶粉在室温封闭滤膜15分钟。迅速地冲洗硝酸纤维素,并加入IgG-HRP抗体(1:10,000)。将所述印迹在轻轻摇动下在4℃孵育过夜。在孵育以后,在TBS中洗涤所述印迹3次,每次10分钟,然后迅速地在H2O中冲洗。使用商购可得的化学发光底物试剂(Roche LumiLight),使所述印迹显影,并使用ImageQuant TL仪器和软件(GE Healthcare)捕获信号。纯化的B7R1小鼠和人Tandem和Barbell Fc融合蛋白在蛋白质印迹和考马斯染色的凝胶上作为2条带出现:在160 kDa附近(在非还原条件下)和在64 kDa附近(在还原条件下),这提示,形成预期的糖基化的二聚形式。所述蛋白具有正确的NH2末端、正确的氨基酸组成,并使用SEC MALS确定地表征了质量和糖基化百分比。
实施例10
B7R1融合蛋白的动态光散射(DLS) 分析
使用平板读数器形式的DynaPro Plus仪器(Wyatt Technology, Santa Barbara,CA),使用动态光散射(DLS),监测人和鼠B7R1融合构建体在21℃、5℃和37℃的短期稳定性和聚集性。作为对比鼠和人形式的这些初步研究的结果,进行其它实验,以便将突变的人barbell和tandem构建体与它们的原始人对应物进行稳定性对比。
1. 关于分析技术的背景
DLS测量散射光强度在微秒时间尺度内的时间依赖性波动。这些强度波动是由布朗运动造成的颗粒在溶液中扩散的结果,并且扩散速率与它们的大小相关。获取DLS原始数据,作为自相关函数图,从该图得到扩散系数(Dt)。当存在多种种类(例如,单体和更大的寡聚体或聚集体)时,在通过Dynamics软件(6.0版, Wyatt Technology)解卷积以后,看到扩散系数的分布。然后使用Stokes-Einstein关系式(其描述了扩散速率和溶液中的颗粒大小之间的理论关系),通过所述软件衍生出流体动力学半径(或Stokes半径)Rh。
DLS对非常少量的大物质极端灵敏,且可以用于可靠地量化最低至总样品的0.01%的蛋白聚集体。
2. 分析参数的描述
样品制备
从-80℃贮存库取出样品,在室温融化30分钟,用于在1 mg/mL的分析,或进行在10K MWCO Amicon单元中的离心浓缩步骤,用于25 mg/mL分析。然后将200 μL等分试样转移至1.5 mL微量离心管,并在Eppendorf台式离心机中在14K rpm在环境温度离心5分钟。离心以后,将20 μL样品转移至Corning 384孔玻璃底平板,用于一式三份地扫描每个样品。将单滴硅油覆盖物加入每个孔中,在分析期间保持。仅在原始制剂缓冲液中试验两种样品浓度。
分析参数
以每个15秒的组在每个孔上收集数据,持续60秒。在室温孵育2小时(就21℃读出而言)、在5℃孵育过夜和在37℃孵育2小时以后,扫描样品。还用新鲜制备的样品进行了在37℃的单独时程实验,以监测在更高的温度随时间的聚集。
3. 结果的总结
DLS大小分布分类的关键
术语“单模态”和“多模态”描述检测到的尖(bins)(或峰)的数目。术语“单分散”和“多分散”描述在每个尖内的种类大小的变异性。例如,样品归类为单模态/多分散= 检测到一个尖,其含有超过一种密切相关的种类,诸如单体/二聚体/三聚体。
在鼠和人形式之间观察到的差异
纯化的鼠B7R1的原始形式的分析表明,所有4种构型,即,Fc (SEQ ID NO:34)、VASP (SEQ ID NO:35)、barbell (SEQ ID NO:10残基26-489;构建体1863)和tandem (SEQID NO:12残基26-504或26-503;构建体1864)形式,在所有3个温度都是稳定的。在37℃没有观察到不稳定性。相反,barbell (SEQ ID NO:6残基22-498;构建体1812)和tandem (SEQID NO:8残基22-513或22-512;构建体1914)分子的人形式在37℃表现出显著无序的聚集和不稳定性,人Fc (B7R1-Fc5;SEQ ID NO:79)和VASP (B7R1-VASP;SEQ ID NO:81)构建体也是如此。在2小时内观察到该聚集,且不是可逆的,这提示,所述结合/聚集已经通过解折叠途径进行。在低和高浓度时观察到人B7R1在37℃的不稳定性,且在分析的批次之间是一致的。
人barbell和tandem形式的突变的影响
还使用DLS,分析了人B7R1 barbell和tandem分子的突变形式:B7R1[G25-P141][C69Y]-Barbell (SEQ ID NO:18残基23-493;参见实施例7)和B7R1[G25-P141][C69Y]-Tandem (SEQ ID NO:20残基23-508或23-507;参见实施例8)。在所有3个温度收集数据,包括在37℃的额外时程研究。
在1 mg/mL,两种突变形式表现出在21℃和5℃的短期稳定性,仅观察到少量多聚化,正如平均半径的稍微增加所指示的。在37℃在T=4小时时观察到59 nm半径的大种类,但是是样品的少量组分(按重量计算,0.1%),且在冷却至环境温度以后可逆地解离。
在25 mg/mL的更高浓度,两种形式具有稍微更大的平均半径和增加的多分散性,指示小的密切相关的种类(诸如二聚体/三聚体)的量的轻微增加,但是样品保持单模态和单分散(即,检测到一个尖),且没有HMW聚集体。
在这2种形式中,认为barbell稍微更稳定*,但是,任一种样品都没有在37℃在短期内表现出任何显著的不稳定性,直到T=18小时最终测量。
结论
在低和高浓度,B7R1[G25-P141][C69Y]-Barbell和B7R1[G25-P141][C69Y]-Tandem都在37℃表现出希望的稳定性增加,彻底改善了用原始的人构建体观察到的聚集问题。
实施例11
人B7R1构建体的结合测定
使用已知的PVR (脊髓灰质炎病毒受体; CD155)-B7R1受体-配体配对,建立含有B7R1受体的蛋白的基于细胞的结合测定。使用标准技术,用含有新霉素(G418) 选择标志物的人PVR表达载体,转染P815细胞(鼠肥大细胞瘤细胞系, ATCC)。在G418选择下生长出来以后,通过流式细胞计量术验证hPVR的表达。具有空载体(对照)的P815细胞的平行转染在G418选择下生长出来,并通过流式细胞术验证没有表达hPVR。
试验了4种含有人B7R1受体的蛋白与表达hPVR的P815细胞结合的能力:A1648.1,一种通过胺缀合用AlexaFluor 647直接标记的hB7R1-VASP分子(SEQ ID NO:81);A2648F,一种hB7R1-Fc2二聚体(SEQ ID NO:79);A2751F,一种hB7R1-”Barbell-Fc”蛋白(SEQ IDNO:18残基23-493);A2752F,一种hB7r1-“Tandem Fc”蛋白(SEQ ID NO:20残基23-508或23-507)。后3种用Zenon-PE鼠IgG标记试剂盒(Invitrogen)直接标记。将每孔100,000个表达hPVR的细胞或阴性对照细胞铺板在96-孔U-底平板中。将所述平板在300xg在室温离心5分钟,并除去上清液。将细胞再悬浮于100µl染色介质(PBS, 1% (v/v) FBS, 0.05% (w/v) 叠氮化钠)中,在5µg/mL开始滴定上述蛋白,并用最低至0.06µg的3倍系列稀释物滴定,得到包括零点对照在内的6-点曲线。将蛋白/细胞在冰上孵育1小时,然后用150µl染色介质洗涤细胞,共洗涤3次。在最后一次洗涤以后,将细胞再悬浮于150µl染色介质:Cytofix (BectonDickenson)的1:1混合物中。通过在LSR II仪器(Becton Dickenson)上的流式细胞术分析细胞。一式三份地测定所有数据点。所有含有hB7R1的蛋白表现出与表达hPVR的P815细胞的剂量依赖性的结合。不存在与阴性对照细胞的结合。
实施例12
人B7R1构建体的竞争测定
建立基于细胞的测定法,用于测量含有人B7R1的构建体的竞争性结合。将每孔100,000个以前描述的表达hPVR的P815细胞铺板在96-孔U-底平板中。将所述平板在300xg在室温离心5分钟,并除去上清液。将细胞再悬浮于50µl染色介质(PBS, 1% (v/v) FBS,0.05% (w/v) 叠氮化钠) 中,所述染色介质含有商购可得的“Fc-block”(BectonDickenson)的1:100稀释物,以使背景结合最小化。在冰上孵育10 min以后,以“3X”浓度加入50µl染色介质,所述染色介质含有一定滴度的含有人B7R1的蛋白,所述滴度是从30µg/mL低至0.042µg/mL的滴度。在该加入以后,立即将50µl含有3µg/mL A1476-Alexa Fluor 647(通过胺缀合用Alexa Fluor 647直接标记的hB7r1-Fc2)的染色介质加入每个孔中,通过轻轻吸吹进行混合。染色在冰上进行1小时。孵育以后,用150µl染色介质洗涤细胞,共洗涤3次。在最后一次洗涤以后,将细胞再悬浮于150µl染色介质:Cytofix (Becton Dickenson)的1:1混合物中。通过在LSR II仪器(Becton Dickenson)上的流式细胞术分析细胞。一式三份地测定所有数据点,并用GraphPad Prism软件,使用4-参数非线性回归曲线拟合计算EC50。
所有含有hB7R1的蛋白都表现出对标记的hB7R1-Fc2结合的剂量依赖性的竞争:B7R1-VASP (SEQ ID NO:81);B7R1-Fc2二聚体(SEQ ID NO:79);B7R1[G25-P141][C69Y]-Barbell (SEQ ID NO:18残基23-493);和B7R1[G25-P141][C69Y]-Tandem (SEQ ID NO:20残基23-508或23-507)。不存在与hPVR表达阴性的“WT”P815细胞的结合。得自2种不同实验的IC50显示在下面的表4中。
表4:B7R1-Fc2与PVR P815细胞的结合的竞争
实施例13
人B7R1 Tandem和Barbell蛋白的生物活性
MHC分子和由抗原呈递细胞(APC)呈递的外来肽与T细胞抗原受体(TCR)的参与,通常会活化T细胞。专职APC也表达许多共刺激性分子,所述分子与在T细胞上的其它受体参与,并促进活化。通过Fc-受体表达细胞将抗体“呈递”给TCR/CD3复合物,可以模仿该过程。然后通过转染Fc-受体表达细胞来提供选择的分子,则可以在某种程度上控制共刺激性分子的呈递。我们使用小鼠肥大细胞瘤细胞系P815,其表达Fc-受体并有效地交联抗-CD3抗体。野生型P815细胞加上抗-CD3抗体可以刺激人T细胞,但是如果P815细胞也表达PVR(CD155),则会增强刺激的水平,因为PVR会参与在T细胞上的CD226 (DNAM-1),从而递送共刺激信号。可溶性B7R1会抑制该相互作用,并阻断CD226介导的共刺激信号。
通过阴性选择(Pan T细胞分离试剂盒, Miltenyi Biotec, Auburn, CA),从外周血中分离人T-细胞,并用CFSE (Invitrogen, Carlsbad, CA)标记。以100,000细胞/孔,将T-细胞铺板在96孔板的每个孔内的生长培养基(RPMI 1640培养基, L-谷氨酰胺, 10%FBS, NEAA, HEPES, Pen-Strep; Invitrogen, Carlsbad, CA)中。在含有和不含有下述额外试剂的情况下一式三份地建立孔:在50,000细胞/孔的P815细胞,或在50,000细胞/孔的用全长人PVR转染且稳定表达所述全长人PVR的P815细胞(P815/PVR),在50 ng/ml的抗-CD3(BD Bioscience, San Diego, CA),在0.6-5 μg/ml的B7R1融合蛋白。在该系统中,对于T细胞,在P815细胞上的Fc-受体交联激动性的抗-CD3 Mabs,以通过T细胞受体(TCR)提供初期的次最佳的刺激,并且PVR通过CD226的参与进行共刺激。可溶性B7R1的加入,将通过结合PVR和阻断CD226活化来抑制共刺激。将样品在37℃孵育4天,收获,并按照典型染色方案,用荧光染料缀合的抗-CD4和抗-CD8 (BD Bioscience, San Diego, CA)染色。通过流式细胞术(LSRII, BD Bioscience, San Diego, CA)分析T细胞,并通过CFSE稀释测量细胞增殖。通过选通(gating)这些特定群体,独立地监测对CD4和CD8 T细胞的影响。
在该实施例中,试验了所述蛋白的2种四聚形式,并与Fc-二聚蛋白进行了对比,所述Fc-二聚蛋白包括下述蛋白批次:
B7r1(G25-P141) C69Y Fc5 barbell (SEQ ID NO:18残基23-493);在本文中也称作B7R1[G25-P141][C69Y]-Barbell;参见实施例7);
B7r1(G25-P141) C69Y Fc5 tandem (SEQ ID NO:20残基23-508或23-507);在本文中也称作B7R1[G25-P141][C69Y]-tandem;参见实施例8);和
B7r1(G25-P141) C69Y Fc5 (二价二聚体) (SEQ ID NO:80)。
图3A-3F指示了通过降低的增殖活性测得的、由可溶性B7R1蛋白诱导的T细胞抑制水平。B7R1-Fc5蛋白具有相对微小的抑制。相比而言,就所有3种血液供体而言,和就CD4+和CD8+ T细胞类型而言,tandem和barbell蛋白都在所有剂量范围内诱导显著的抑制,其中tandem表现出比barbell稍微更高的活性。这指示,这些蛋白是T细胞体外增殖的有效抑制剂,且优于二聚形式,并进一步提示,它们在临床场合也将优于二价的二聚体。
实施例14
可溶性的B7R1受体(Barbell构建体)在多发性硬化的小鼠实验性变应性脑脊髓炎(EAE) 模型中降低疾病发病率和进展
A) 小鼠变应性脑脊髓炎(EAE) 模型
为了研究机理和评价对多发性硬化的潜在治疗效果,通常使用实验性自身免疫性脑脊髓炎(EAE)的动物模型。就慢性进行性EAE模型而言,在第0天,用在完全弗氏佐剂中乳化的髓磷脂少突神经胶质细胞糖蛋白(MOG) 35-55肽,皮下地免疫8-10周龄雌性C57BL/6小鼠(Charles River Laboratories),随后在第0天腹膜内递送百日咳毒素,并在第2天静脉内递送百日咳毒素。在约8-23天内,动物开始表现出该模型的特征性的重量减轻和麻痹症状。如下面详述地,通过测量它们的体重,并将临床评分(0-8) 分配给每只小鼠,每天评价小鼠的疾病程度。在免疫的、但是在其它方面没有处理的小鼠中的疾病症状的典型模式是重量减轻和麻痹之一。
在第-1天开始,将可溶性的B7R1受体barbell构建体(mB7R1-Barbell;SEQ ID NO:10的残基26-489)、二聚的鼠Fc2构建体(SEQ ID NO:34)、VASP构建体(SEQ ID NO:35)或媒介(PBS) 施用给小鼠。每隔1天递送治疗作为腹膜内注射,以每只小鼠每剂150 μg,施用每种B7R1分子。还使用类似的给药方案或其它施用途径递送它们。
B) 监测疾病
在MOG35-55免疫接种以后约8-23天,动物可以开始表现出麻痹和重量减轻的迹象。大多数动物在免疫接种的11–17天内显现症状,但是有些可以比这更早或更晚地表现出症状。
每天对所有动物进行观察、称重和分配临床评分,以评估疾病的状态。
C) 临床评分
0 = 正常的;健康的。
1 = 轻微的尾巴虚弱(尾巴尖不再卷曲)
2 = 尾巴麻痹(不能保持尾巴直立)
3 = 尾巴麻痹和轻度蹒跚
4 = 尾巴麻痹和严重蹒跚
5 = 尾巴麻痹和一肢麻痹
6 = 尾巴麻痹和任意2肢麻痹
7 = 四肢轻瘫(所有4肢麻痹)
8 = 垂死或死亡
在实验过程中和结束时收集血液,以监测细胞因子的血清水平和疾病的其它介导因子的水平。在安乐死时,收集血液制备血清。
D) 结果
通过疾病严重性随时间的显著(p<0.05)降低,表征接受mB7R1-Barbell的小鼠组(每组n = 10-12),所述显著降低表现为,通过重复测量双因素方差分析(ANOVA) 得出的与媒介(PBS)治疗的小鼠相比临床评分的显著(p<0.05)降低(参见图4)。就用二聚的鼠Fc2或VASP构建体治疗的小鼠组而言,存在在一定程度上被保护免于疾病的趋势,表现为疾病进展的延迟。但是,通过双因素方差分析,这2组和媒介治疗组之间的差异没有显著不同。当分析为随时间的疾病评分总和(类似于曲线下面积计算或“疾病暴露”)时,与用PBS治疗的小鼠相比,用B7R1-Barbell构建体治疗的小鼠具有显著降低的平均“疾病暴露”指数(累积评分下降了约2.3倍;p < 0.05)。与PBS治疗组相比,用二聚的鼠Fc2或VASP构建体各自治疗的小鼠组具有降低了约25%的累积评分,但是差异不是统计上不同的。用B7R1-Barbell构建体和二聚的鼠Fc2构建体各自治疗的小鼠具有更低的疾病发作率,使得约25%的B7R1-Barbell-和二聚的鼠Fc2治疗组的小鼠被保护免于疾病,约17% 的VASP治疗的小鼠被保护免于疾病,且仅约8%的PBS治疗的小鼠受到保护。此外,就接受B7R1-Barbell的小鼠而言,疾病发作的平均天数是19天,二聚的鼠Fc2治疗的小鼠是17.6天,VASP治疗的小鼠是16.5天,而PBS治疗的小鼠的疾病发作平均天数则更短(13.7天),这表明,可溶性B7R1分子、特别是B7R1-Barbell治疗会延迟疾病发作。
这些结果一起证实,B7R1-Barbell的体内施用可有效地减少在EAE(一种人多发性硬化模型)中的疾病发作和严重性。存在用其它可溶性B7R1分子观察到效力的趋势,尽管Barbell构建体是在该实验中表现出统计上显著差异的唯一可溶性受体。这些结果提示,可溶性B7R1 barbell构建体可有效地治疗人多发性硬化。
实施例15
B7R1 Tandem构建体在作为多发性硬化模型的复发缓解型(RR)小鼠实验性变应性脑脊髓炎(EAE) 中降低疾病发病率和进展
A) 小鼠变应性脑脊髓炎(EAE) 模型
为了研究机理和评价对多发性硬化的潜在治疗效果,通常使用实验性自身免疫性脑脊髓炎(EAE)的动物模型。就复发缓解型EAE模型而言,用在完全弗氏佐剂中乳化蛋白脂质肽(PLP),皮下地免疫9-10周龄雌性SJL小鼠(Jackson或Charles River Labs),没有静脉内递送百日咳毒素。在约14-18天内,动物开始表现出该模型的特征性的重量减轻和麻痹症状。如下面详述地,通过测量它们的体重,并将临床评分(0-8)分配给每只小鼠,每天评价小鼠的疾病程度。在免疫的、但是在其它方面没有处理的小鼠中的疾病症状的典型模式是重量减轻和麻痹之一,继之以疾病症状减轻阶段,和随后的疾病症状复发。疾病症状的复发和减轻的模式相继发生(这也存在于具有这类多发性硬化的人类中),称作复发缓解型疾病。
在治疗给药方案中施用可溶性的B7R1受体tandem构建体(mB7R1-Tandem;SEQ IDNO:12的残基26-504或26-503)、可溶性的B7R1受体barbell构建体(mB7R1-Barbell;SEQ IDNO:10的残基26-489)、媒介(PBS)、或临床上有关的阳性对照(鼠-特异性的CTLA4-Ig),使得它们在已经度过它们的第一个疾病高峰以后第二天开始治疗。每隔1天递送治疗作为腹膜内注射,以每只小鼠每剂150 μg,施用mB7R1-Tandem、mB7R1-Barbell和mCTLA4-Ig分子。还使用类似的给药方案或其它施用途径递送它们。
B) 监测疾病
在PLP免疫接种以后约8-23天,动物可以开始表现出麻痹和重量减轻的迹象。大多数动物在该实验中在免疫接种的14–18天内显现症状。
每天对所有动物进行观察、称重和分配临床评分,以评估疾病的状态。
C) 临床评分
0 = 正常的;健康的。
1 = 轻微的尾巴虚弱(尾巴尖不再卷曲)
2 = 尾巴麻痹(不能保持尾巴直立)
3 = 尾巴麻痹和轻度蹒跚
4 = 尾巴麻痹和严重蹒跚
5 = 尾巴麻痹和一肢麻痹
6 = 尾巴麻痹和任意2肢麻痹
7 = 四肢轻瘫(所有4肢麻痹)
8 = 垂死或死亡。
D) 结果
通过疾病严重性显著(p<0.05)降低进入缓解期,表征用B7R1-Tandem治疗的小鼠组(每组n = 12-13),所述缓解期表现为,与媒介(PBS)治疗的小鼠相比,临床评分和体重减轻的显著(p<0.05)降低。此外,与媒介(PBS)治疗的小鼠相比,用B7R1-Tandem治疗的小鼠具有明显更低的(p<0.05) 疾病发生。这是非常重要的发现,因为疾病复发是该疾病模型和人类的复发缓解型MS的标志。用B7R1-Barbell分子治疗的小鼠组具有与用阳性对照mCTLA4-Ig治疗的小鼠组类似的疾病评分和复发率。与PBS治疗的小鼠组相比,B7R1-Barbell和mCTLA4-Ig治疗的小鼠组都没有显著不同的疾病评分或复发率。
这些结果一起证实,B7R1-Tandem的体内施用可有效地减少在PLP EAE(一种人复发缓解型多发性硬化模型)中的疾病发作和严重性。这些结果提示,可溶性B7R1-Tandem构建体可有效地治疗人多发性硬化。
实施例16
B7R1-Tandem在T-细胞过继转移结肠炎和银屑病的小鼠模型中降低疾病发生和进展
T-细胞过继转移结肠炎模型
原初T细胞向次要组织相容性错配的或同基因的免疫受损小鼠中的过继转移,会导致结肠炎(Leach等人, 1996; Powrie等人, 1997) 以及类似于银屑病的皮肤病变(Schon等人, 1997; Davenport等人, 2002)的发展。从BALB/C或B10.D2小鼠向免疫受损的C.B-17 SCID小鼠中移植低至20万CD4+ CD25-T细胞,会导致重量减轻、潜血检测阳性的粪便和皮肤病变的发展。在这些小鼠中的症状随集落不同而变化。
该结肠炎/银屑病模型与人克罗恩氏病和银屑病具有一些相似性,且已经被广泛地用于试验用于人类的这些疾病的治疗剂的效力。就该实验而言,分别从JacksonLaboratories或Charles River Laboratories得到小鼠(5只B10.D2雌性小鼠供体;20只C.B-17 SCID雌性受体)。从5只B10.D2小鼠收集脾脏。使用本领域已知的标准方法,从合并的脾脏分离CD4+ CD25-T-细胞。通过流式细胞术,评价T-细胞的纯度。
通过静脉内注射,C.B-17 SCID小鼠接受5x105-10x105个得自脾脏的CD4+ CD25-T-细胞。每周对所有小鼠称重至少5次,并仔细地观察重量减轻。每周至少1天进行临床结肠炎评分(粪便稠度和粪便带血)。还每周至少5天监测小鼠,并分配银屑病的迹象和程度的评分(毛发脱落、搔抓和斑秃)。
在第0天(细胞转移当日)开始,给小鼠施用可溶性的B7R1受体tandem构建体(B7R1-Tandem)或媒介(PBS)。每隔1天递送治疗作为腹膜内注射,以每只小鼠每剂150 μg,施用B7R1-Tandem。还使用类似的给药方案或其它施用途径递送它们。
在研究结束时,将组织样品(例如肠和皮肤)分别用于检测结肠炎和银屑病的组织学证据,并收集血清用于分析细胞因子和趋化因子水平。
结果
通过疾病严重性的显著(p<0.05)降低,表征接受B7R1-Tandem的小鼠组(每组n=10),所述显著降低表现为,与媒介(PBS)治疗的小鼠相比,结肠炎和银屑病的临床评分和体重减轻的显著(p<0.05)降低。此外,与媒介(PBS)治疗的小鼠相比,用B7R1-Tandem治疗的小鼠具有更少的结肠炎和银屑病病理学/组织学证据。
这些结果一起证实,B7R1-Tandem的体内施用可以在鼠T细胞转移模型中有效地减轻结肠炎和银屑病,并提示,该可溶性受体可以有效地治疗人炎性肠病和/或银屑病。
实施例17
B7R1可溶性受体(Barbell和Tandem构建体)在小鼠胶原诱导的关节炎(CIA) 模型中降低疾病发生和进展
A) 小鼠胶原诱导的关节炎(CIA) 模型
将10周龄雄性DBA/1J小鼠(Jackson Labs)分成3组,每组15只小鼠,指定用于预防性施用PBS、B7R1 barbell蛋白或B7R1 tandem蛋白。在第-21天,给所有动物施用50-100 μL在完全弗氏佐剂(由Chondrex, Redmond, WA制备)中配制的1mg/mL鸡II型胶原的皮内尾巴注射,并在3周后第0天,给它们施用相同的注射,除了在不完全弗氏佐剂中制备。在第-1天开始,每隔1天施用作为腹膜内注射的B7R1-Barbell (mB7R1-Barbell;SEQ ID NO:10的残基26-489)、B7R1-Tandem (mB7R1-Tandem;SEQ ID NO:12的残基26-504或26-503)、B7R1-VASP (mB7R1-VASP;SEQ ID NO:35)或媒介(PBS),持续3周。试验组接受每只动物每剂150 μg B7R1-Barbell或B7R1-Tandem蛋白,对照组接受媒介对照PBS (Life Technologies,Rockville, MD)。在第二次胶原注射以后,动物开始表现出关节炎的症状,大多数动物在1-2周内发展出炎症。通过使用卡尺测量爪厚度,并通过给每只爪分配临床评分(0-3)(参见下文),评价每只爪中的疾病程度。
B) 监测疾病
在第二次胶原注射以后不久,动物可以开始表现出爪炎症迹象,并且一些动物甚至在第二次胶原注射之前可以开始具有趾炎症的迹象。大多数动物在增强注射的1-2周内显现关节炎,但是有些可能需要更长的时间段。在该模型中的疾病发病率通常是90-100%,且在使用60只动物的研究中,通常观察到0-5只无应答者(在观察6周以后确定)。
每天观察所有动物,以评估它们的爪子的疾病状态,这通过将定性临床评分分配给每只爪子来实现。每天,根据爪子的临床疾病状态,对每只动物的4只爪子评分。为了确定临床评分,可以认为爪子具有3个区域:趾、爪子本身(前足或后足)以及腕或踝关节。观察与这些区域有关的炎症的程度和严重性,包括:观察每个趾的肿胀;撕裂的指甲或趾发红;记录任一只爪子中的任何水肿或发红证据;记录腱或骨的细微解剖学分界的任何消失;评价腕或踝的任何水肿或发红;和记录炎症是否向近端向上延伸至腿。1、2或3的爪子评分首先是基于严重性的总体印象,其次是基于多少区域受到影响。下面显示了用于临床评分的标度。
C) 临床评分
0 = 正常
0.5 = 一个或多个趾受到影响,但是仅趾发炎
1 = 影响爪子(1个区域)的轻度炎症,且可以包括一个或多个趾
2 = 在爪子中的中等炎症,且可以包括一些趾和/或腕/踝(2个区域)
3 = 在爪子、腕/踝和一些或所有趾(3个区域)中的严重炎症。
在实验结束时收集血液,以监测抗-胶原抗体的血清水平、以及血清趋化因子和细胞因子水平。
通过与接受PBS的小鼠相比随着实验进程显著减少的关节炎和爪炎症(p<0.05)(参见图5),表征接受B7R1-Barbell和B7R1-Tandem分子的小鼠组。此外,与接受B7R1-VASP蛋白的小鼠相比,接受B7R1-Tandem的小鼠倾向于随着实验进程具有甚至更少的关节炎和爪炎症(p=0.09)。与PBS治疗的小鼠相比,在用B7R1-Barbell和B7R1-Tandem分子治疗的小鼠中,IL-6和趋化因子MCP-1、IP-1和KC的血清水平更低。抗-胶原抗体的血清水平在各组之间没有显著差异。这些结果指示,B7R1-Barbell和B7R1-Tandem可以减轻炎症以及与该关节炎模型有关的疾病进展,并提示,B7R1可溶性受体的B7R1 barbell或tandem构建体可以有效地治疗人关节炎。
从前述内容可以理解,尽管本文中已经描述了本发明的具体实施方案用于例证目的,可以做出各种改进,而不偏离本发明的精神和范围。因此,本发明仅受所附权利要求限制。在本文中引用的所有出版物、专利和专利申请都特此通过引用以它们的整体并入用于所有目的。
序列表
<110> Levin, Steven D.
Ostrander, Craig D.
Moore, Margaret D.
<120> 二聚VSTM3融合蛋白和相关的组合物和方法
<130> Z01001 PCT
<150> 61/352873
<151> 06/09/10
<160> 81
<170> PatentIn 3.5版
<210> 1
<211> 735
<212> DNA
<213> 智人
<220>
<221> misc_feature
<222> (349)..(349)
<223> n是a、c、g或t
<220>
<221> misc_feature
<222> (359)..(359)
<223> n是a或g
<400> 1
atgcgctggt gtctcctcct gatctgggcc caggggctga ggcaggctcc cctcgcctca 60
ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct 120
atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt caactgggag 180
cagcaggacc agcttctggc catttgtaat gctgacttgg ggtggcacat ctccccatcc 240
ttcaaggatc gagtggcccc aggtcccggc ctgggcctca ccctccagtc gctgaccgtg 300
aacgatacag gggagtactt ctgcatctat cacacctacc ctgatgggnc gtacactggg 360
agaatcttcc tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 420
ccattgcttg gagccatggc cgcgacgctg gtggtcatct gcacagcagt catcgtggtg 480
gtcgcgttga ctagaaagaa gaaagccctc agaatccatt ctgtggaagg tgacctcagg 540
agaaaatcag ctggacagga ggaatggagc cccagtgctc cctcaccccc aggaagctgt 600
gtccaggcag aagctgcacc tgctgggctc tgtggagagc agcggggaga ggactgtgcc 660
gagctgcatg actacttcaa tgtcctgagt tacagaagcc tgggtaactg cagcttcttc 720
acagagactg gttag 735
<210> 2
<211> 244
<212> PRT
<213> 智人
<220>
<221> misc_feature
<222> (117)..(117)
<223> Xaa是Thr或Ala
<400> 2
Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala
1 5 10 15
Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
20 25 30
Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser
35 40 45
Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
50 55 60
Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser
65 70 75 80
Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln
85 90 95
Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
100 105 110
Tyr Pro Asp Gly Xaa Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu
115 120 125
Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly
130 135 140
Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val
145 150 155 160
Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu
165 170 175
Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser
180 185 190
Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala
195 200 205
Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp
210 215 220
Tyr Phe Asn Val Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe
225 230 235 240
Thr Glu Thr Gly
<210> 3
<211> 726
<212> DNA
<213> 小家鼠
<400> 3
atgcatggct ggctgctcct ggtctgggtc caggggctga tacaggctgc cttcctcgct 60
acaggagcca cagcaggcac gatagataca aagaggaaca tctctgcaga ggaaggtggc 120
tctgtcatct tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca 240
gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca gtctctgaca 300
atgaatgaca cgggagagta cttctgtacc tatcatacgt atcctggtgg gatttacaag 360
gggagaatat tcctgaaggt ccaagaaagc tcagtggctc agttccagac tgccccgctt 420
ggaggaacca tggctgctgt gctgggactc atttgcttaa tggtcacagg agtgactgta 480
ctggctagaa agaagtctat tagaatgcat tctatagaaa gtggccttgg gagaacagaa 540
gcggagccac aggaatggaa cctgaggagt ctctcatccc ctggaagccc tgtccagaca 600
caaactgccc ctgctggtcc ctgtggagag caggcagaag atgactatgc tgacccacag 660
gaatacttta atgtcctgag ctacagaagc ctagagagct tcattgctgt atcgaagact 720
ggctaa 726
<210> 4
<211> 241
<212> PRT
<213> 小家鼠
<400> 4
Met His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala
1 5 10 15
Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg
20 25 30
Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe
35 40 45
Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp
50 55 60
Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser
65 70 75 80
Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe
85 90 95
Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His
100 105 110
Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln
115 120 125
Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Pro Leu Gly Gly Thr Met
130 135 140
Ala Ala Val Leu Gly Leu Ile Cys Leu Met Val Thr Gly Val Thr Val
145 150 155 160
Leu Ala Arg Lys Lys Ser Ile Arg Met His Ser Ile Glu Ser Gly Leu
165 170 175
Gly Arg Thr Glu Ala Glu Pro Gln Glu Trp Asn Leu Arg Ser Leu Ser
180 185 190
Ser Pro Gly Ser Pro Val Gln Thr Gln Thr Ala Pro Ala Gly Pro Cys
195 200 205
Gly Glu Gln Ala Glu Asp Asp Tyr Ala Asp Pro Gln Glu Tyr Phe Asn
210 215 220
Val Leu Ser Tyr Arg Ser Leu Glu Ser Phe Ile Ala Val Ser Lys Thr
225 230 235 240
Gly
<210> 5
<211> 1494
<212> DNA
<213> 人工序列
<220>
<223> B7R1 Fc5 Barbell
<400> 5
atgcgctggt gtctcctcct gatctgggcc caggggctga ggcaggctcc cctcgcctca 60
ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct 120
atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt caactgggag 180
cagcaggacc agcttctggc catttgtaat gctgacttgg ggtggcacat ctccccatcc 240
ttcaaggatc gagtggcccc aggtcccggc ctgggcctca ccctccagtc gctgaccgtg 300
aacgatacag gggagtactt ctgcatctat cacacctacc ctgatgggac gtacactggg 360
agaatcttcc tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 420
ccagagccca aatcttcaga caaaactcac acatgcccac cgtgcccagc acctgaagcc 480
gagggggcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 540
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 600
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 660
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 720
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccatcctc catcgagaaa 780
accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 840
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 900
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 960
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag1020
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac1080
cactacacgc agaagagcct ctccctgtct ccgggtaaag gtggtgggtc cggaatgatg1140
acaggcacaa tagaaacaac ggggaacatt tctgcagaga aaggtggctc tatcatctta1200
caatgtcacc tctcctccac cacggcacaa gtgacccagg tcaactggga gcagcaggac1260
cagcttctgg ccatttgtaa tgctgacttg gggtggcaca tctccccatc cttcaaggat1320
cgagtggccc caggtcccgg cctgggcctc accctccagt cgctgaccgt gaacgataca1380
ggggagtact tctgcatcta tcacacctac cctgatggga cgtacactgg gagaatcttc1440
ctggaggtcc tagaaagctc agtggctgag cacggtgcca ggttccagat tcca 1494
<210> 6
<211> 498
<212> PRT
<213> 人工序列
<220>
<223> B7R1 Fc5 Barbell
<400> 6
Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala
1 5 10 15
Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
20 25 30
Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser
35 40 45
Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
50 55 60
Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser
65 70 75 80
Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln
85 90 95
Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
100 105 110
Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu
115 120 125
Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys
130 135 140
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
145 150 155 160
Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
165 170 175
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
180 185 190
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
195 200 205
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
210 215 220
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
225 230 235 240
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser
245 250 255
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
260 265 270
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
275 280 285
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
290 295 300
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
305 310 315 320
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
325 330 335
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
340 345 350
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
355 360 365
Leu Ser Pro Gly Lys Gly Gly Gly Ser Gly Met Met Thr Gly Thr Ile
370 375 380
Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu
385 390 395 400
Gln Cys His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp
405 410 415
Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp
420 425 430
His Ile Ser Pro Ser Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu
435 440 445
Gly Leu Thr Leu Gln Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe
450 455 460
Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe
465 470 475 480
Leu Glu Val Leu Glu Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln
485 490 495
Ile Pro
<210> 7
<211> 1539
<212> DNA
<213> 人工序列
<220>
<223> B7R1 Fc5 Tandem
<400> 7
atgcgctggt gtctcctcct gatctgggcc caggggctga ggcaggctcc cctcgcctca 60
ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct 120
atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt caactgggag 180
cagcaggacc agcttctggc catttgtaat gctgacttgg ggtggcacat ctccccatcc 240
ttcaaggatc gagtggcccc aggtcccggc ctgggcctca ccctccagtc gctgaccgtg 300
aacgatacag gggagtactt ctgcatctat cacacctacc ctgatgggac gtacactggg 360
agaatcttcc tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 420
ccaggcggtt cgggctcagg atcgggaggt tcaggttccg gtggttctgg tggtgggtcc 480
ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct 540
atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt caactgggag 600
cagcaggacc agcttctggc catttgtaat gctgacttgg ggtggcacat ctccccatcc 660
ttcaaggatc gagtggcccc aggtcccggc ctgggcctca ccctccagtc gctgaccgtg 720
aacgatacag gggagtactt ctgcatctat cacacctacc ctgatgggac gtacactggg 780
agaatcttcc tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 840
ccagagccca aatcttcaga caaaactcac acatgcccac cgtgcccagc acctgaagcc 900
gagggggcac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 960
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag1020
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag1080
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg1140
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccatcctc catcgagaaa1200
accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc1260
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc1320
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg1380
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag1440
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac1500
cactacacgc agaagagcct ctccctgtct ccgggtaaa 1539
<210> 8
<211> 513
<212> PRT
<213> 人工序列
<220>
<223> B7R1 Fc5 Tandem
<400> 8
Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala
1 5 10 15
Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn
20 25 30
Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser
35 40 45
Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
50 55 60
Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser
65 70 75 80
Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln
85 90 95
Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
100 105 110
Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu
115 120 125
Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Gly Gly Ser
130 135 140
Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Ser
145 150 155 160
Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu
165 170 175
Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala
180 185 190
Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile
195 200 205
Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg
210 215 220
Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val
225 230 235 240
Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly
245 250 255
Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala
260 265 270
Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp Lys
275 280 285
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro
290 295 300
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
305 310 315 320
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
325 330 335
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
340 345 350
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
355 360 365
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
370 375 380
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys
385 390 395 400
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
405 410 415
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
420 425 430
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
435 440 445
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
450 455 460
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
465 470 475 480
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
485 490 495
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
500 505 510
Lys
<210> 9
<211> 1467
<212> DNA
<213> 人工序列
<220>
<223> mB7R1 mFc2 Barbell
<400> 9
atgcatggct ggctgctcct ggtctgggtc caggggctga tacaggctgc cttcctcgct 60
acaggagcca cagcaggcac gatagataca aagaggaaca tctctgcaga ggaaggtggc 120
tctgtcatct tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca 240
gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca gtctctgaca 300
atgaatgaca cgggagagta cttctgtacc tatcatacgt atcctggtgg gatttacaag 360
gggagaatat tcctgaaggt ccaagaaagc tcagtggctc agttccagac tgccgagccc 420
agatctccca caatcaagcc ctgtcctcca tgcaaatgcc cagcacctaa cctcgagggt 480
ggaccatccg tcttcatctt ccctccaaag atcaaggatg tactcatgat ctccctgagc 540
cccatagtca catgtgtggt ggtggatgtg agcgaggatg acccagatgt ccagatcagc 600
tggtttgtga acaacgtgga agtacacaca gctcagacac aaacccatag agaggattac 660
aacagtactc tccgggtggt cagtgccctc cccatccagc accaggactg gatgagtggc 720
aaagctttcg catgcgcggt caacaacaaa gacctcccag cgcccatcga gagaaccatc 780
tcaaaaccca aagggtcagt aagagctcca caggtatatg tcttgcctcc accagaagaa 840
gagatgacta agaaacaggt cactctgacc tgcatggtca cagacttcat gcctgaagac 900
atttacgtgg agtggaccaa caacgggaaa acagagctaa actacaagaa cactgaacca 960
gtcctggact ctgatggttc ttacttcatg tacagcaagc tgagagtgga aaagaagaac1020
tgggtggaaa gaaatagcta ctcctgttca gtggtccacg agggtctgca caatcaccac1080
acgactaaga gcttctcccg gactccgggt aaaggtggtg ggtccggagg cacgatagat1140
acaaagagga acatctctgc agaggaaggt ggctctgtca tcttacagtg tcacttctcc1200
tctgacacag ctgaagtgac ccaagtcgac tggaagcagc aggaccagct tctggccatt1260
tatagtgttg acctggggtg gcatgtcgct tcagtcttca gtgatcgggt ggtcccaggc1320
cccagcctag gcctcacctt ccagtctctg acaatgaatg acacgggaga gtacttctgt1380
acctatcata cgtatcctgg tgggatttac aaggggagaa tattcctgaa ggtccaagaa1440
agctcagtgg ctcagttcca gactgcc 1467
<210> 10
<211> 489
<212> PRT
<213> 人工序列
<220>
<223> mB7R1 mFc2 Barbell
<400> 10
Met His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala
1 5 10 15
Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg
20 25 30
Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe
35 40 45
Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp
50 55 60
Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser
65 70 75 80
Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe
85 90 95
Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His
100 105 110
Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln
115 120 125
Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Glu Pro Arg Ser Pro Thr
130 135 140
Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Glu Gly
145 150 155 160
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met
165 170 175
Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu
180 185 190
Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val
195 200 205
His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu
210 215 220
Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly
225 230 235 240
Lys Ala Phe Ala Cys Ala Val Asn Asn Lys Asp Leu Pro Ala Pro Ile
245 250 255
Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val
260 265 270
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr
275 280 285
Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu
290 295 300
Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro
305 310 315 320
Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val
325 330 335
Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val
340 345 350
His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr
355 360 365
Pro Gly Lys Gly Gly Gly Ser Gly Gly Thr Ile Asp Thr Lys Arg Asn
370 375 380
Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe Ser
385 390 395 400
Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp Gln
405 410 415
Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser Val
420 425 430
Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe Gln
435 440 445
Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His Thr
450 455 460
Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln Glu
465 470 475 480
Ser Ser Val Ala Gln Phe Gln Thr Ala
485
<210> 11
<211> 1527
<212> DNA
<213> 人工序列
<220>
<223> mB7R1 mFc2 Tandem
<400> 11
atgcatggct ggctgctcct ggtctgggtc caggggctga tacaggctgc cttcctcgct 60
acaggagcca cagcaggcac gatagataca aagaggaaca tctctgcaga ggaaggtggc 120
tctgtcatct tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca 240
gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca gtctctgaca 300
atgaatgaca cgggagagta cttctgtacc tatcatacgt atcctggtgg gatttacaag 360
gggagaatat tcctgaaggt ccaagaaagc tcagtggctc agttccagac tgccggcggt 420
tcgggctcag gatcgggagg ttcaggttcc ggtggttctg gtggtgggtc cggaggcacg 480
atagatacaa agaggaacat ctctgcagag gaaggtggct ctgtcatctt acagtgtcac 540
ttctcctctg acacagctga agtgacccaa gtcgactgga agcagcagga ccagcttctg 600
gccatttata gtgttgacct ggggtggcat gtcgcttcag tcttcagtga tcgggtggtc 660
ccaggcccca gcctaggcct caccttccag tctctgacaa tgaatgacac gggagagtac 720
ttctgtacct atcatacgta tcctggtggg atttacaagg ggagaatatt cctgaaggtc 780
caagaaagct cagtggctca gttccagact gccgagccca gatctcccac aatcaagccc 840
tgtcctccat gcaaatgccc agcacctaac ctcgagggtg gaccatccgt cttcatcttc 900
cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatagtcac atgtgtggtg 960
gtggatgtga gcgaggatga cccagatgtc cagatcagct ggtttgtgaa caacgtggaa1020
gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc1080
agtgccctcc ccatccagca ccaggactgg atgagtggca aagctttcgc atgcgcggtc1140
aacaacaaag acctcccagc gcccatcgag agaaccatct caaaacccaa agggtcagta1200
agagctccac aggtatatgt cttgcctcca ccagaagaag agatgactaa gaaacaggtc1260
actctgacct gcatggtcac agacttcatg cctgaagaca tttacgtgga gtggaccaac1320
aacgggaaaa cagagctaaa ctacaagaac actgaaccag tcctggactc tgatggttct1380
tacttcatgt acagcaagct gagagtggaa aagaagaact gggtggaaag aaatagctac1440
tcctgttcag tggtccacga gggtctgcac aatcaccaca cgactaagag cttctcccgg1500
actccgggta aaggtggtgg gtccgga 1527
<210> 12
<211> 509
<212> PRT
<213> 人工序列
<220>
<223> mB7R1 mFc2 Tandem
<400> 12
Met His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala
1 5 10 15
Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg
20 25 30
Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe
35 40 45
Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp
50 55 60
Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser
65 70 75 80
Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe
85 90 95
Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His
100 105 110
Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln
115 120 125
Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Gly Gly Ser Gly Ser Gly
130 135 140
Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Ser Gly Gly Thr
145 150 155 160
Ile Asp Thr Lys Arg Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile
165 170 175
Leu Gln Cys His Phe Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp
180 185 190
Trp Lys Gln Gln Asp Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly
195 200 205
Trp His Val Ala Ser Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser
210 215 220
Leu Gly Leu Thr Phe Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr
225 230 235 240
Phe Cys Thr Tyr His Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile
245 250 255
Phe Leu Lys Val Gln Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Glu
260 265 270
Pro Arg Ser Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
275 280 285
Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
290 295 300
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
305 310 315 320
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
325 330 335
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
340 345 350
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
355 360 365
Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val Asn Asn Lys Asp
370 375 380
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
385 390 395 400
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
405 410 415
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
420 425 430
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
435 440 445
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
450 455 460
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
465 470 475 480
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
485 490 495
Ser Phe Ser Arg Thr Pro Gly Lys Gly Gly Gly Ser Gly
500 505
<210> 13
<211> 1518
<212> DNA
<213> 人工序列
<220>
<223> B7R1[G25-P141] Fc5 Barbell (otPA前导序列)>
<400> 13
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggcgc cgtcttcgtt 60
tcgctcagcc aggaaatcca tgccgagttg agacgcttcc gtagaggcac aatagaaaca 120
acggggaaca tttctgcaga gaaaggtggc tctatcatct tacaatgtca cctctcctcc 180
accacggcac aagtgaccca ggtcaactgg gagcagcagg accagcttct ggccatttgt 240
aatgctgact tggggtggca catctcccca tccttcaagg atcgagtggc cccaggtccc 300
ggcctgggcc tcaccctcca gtcgctgacc gtgaacgata caggggagta cttctgcatc 360
tatcacacct accctgatgg gacgtacact gggagaatct tcctggaggt cctagaaagc 420
tcagtggctg agcacggtgc caggttccag attccagagc ccaaatcttc agacaaaact 480
cacacatgcc caccgtgccc agcacctgaa gccgaggggg caccgtcagt cttcctcttc 540
cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 600
gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 660
gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 720
agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 780
tccaacaaag ccctcccatc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 840
cgagagccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 900
agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 960
aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc1020
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc1080
tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg1140
tctccgggta aaggtggtgg gtccggaggt acaattgaaa cgacaggcaa tatatctgcc1200
gagaagggtg gctcgatcat attacaatgc cacctctcat ccaccactgc acaagtaacc1260
caggtcaatt gggaacagca ggatcagctg ctggccatct gtaacgctga cttgggatgg1320
cacatttccc catcgttcaa agatcgagtc gccccaggcc ccggcctcgg cctcacactc1380
cagtctctga ccgtcaacga tacaggcgag tacttttgca tatatcacac ttaccctgac1440
gggacctaca ctggtagaat cttcctagag gtcctagaga gctcagtcgc tgagcatggg1500
gctagattcc agatccca 1518
<210> 14
<211> 506
<212> PRT
<213> 人工序列
<220>
<223> B7R1[G25-P141] Fc5 Barbell (otPA前导序列)
<400> 14
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg
20 25 30
Phe Arg Arg Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys
35 40 45
Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln
50 55 60
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys
65 70 75 80
Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val
85 90 95
Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn
100 105 110
Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
115 120 125
Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu
130 135 140
His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp Lys Thr
145 150 155 160
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser
165 170 175
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
180 185 190
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
195 200 205
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
210 215 220
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
225 230 235 240
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
245 250 255
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr
260 265 270
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
275 280 285
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
290 295 300
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
305 310 315 320
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
325 330 335
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
340 345 350
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
355 360 365
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
Gly Gly Gly Ser Gly Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala
385 390 395 400
Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr
405 410 415
Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
420 425 430
Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp
435 440 445
Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr
450 455 460
Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp
465 470 475 480
Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val
485 490 495
Ala Glu His Gly Ala Arg Phe Gln Ile Pro
500 505
<210> 15
<211> 1518
<212> DNA
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5 Barbell (otPA前导序列)
<400> 15
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggcgc cgtcttcgtt 60
tcgctcagcc aggaaatcca tgccgagttg agacgcttcc gtagaggcac aatagaaaca 120
acggggaaca tttctgcaga gaaaggtggc tctatcatct tacaatgtca cctctcctcc 180
accacggcac aagtgaccca ggtcaactgg gagcagcagg accagcttct ggccatttat 240
aatgctgact tggggtggca catctcccca tccttcaagg atcgagtggc cccaggtccc 300
ggcctgggcc tcaccctcca gtcgctgacc gtgaacgata caggggagta cttctgcatc 360
tatcacacct accctgatgg gacgtacact gggagaatct tcctggaggt cctagaaagc 420
tcagtggctg agcacggtgc caggttccag attccagagc ccaaatcttc agacaaaact 480
cacacatgcc caccgtgccc agcacctgaa gccgaggggg caccgtcagt cttcctcttc 540
cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 600
gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 660
gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 720
agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 780
tccaacaaag ccctcccatc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 840
cgagagccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 900
agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 960
aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc1020
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc1080
tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg1140
tctccgggta aaggtggtgg gtccggaggt acaattgaaa caacaggcaa tatatctgcc1200
gagaagggtg gctcgatcat attacaatgc cacctctcat ccaccactgc acaagtaacc1260
caggtcaatt gggaacagca ggatcagctg ctggccatct ataacgctga cttgggatgg1320
cacatttccc catcgttcaa agatcgagtc gccccaggcc ccggcctcgg cctcacactc1380
cagtctctga ccgtcaacga tacaggcgag tacttttgca tatatcacac ttaccctgac1440
gggacctaca ctggtagaat cttcctagag gtcctagaga gctcagtcgc tgagcatggg1500
gctagattcc agatccca 1518
<210> 16
<211> 506
<212> PRT
<213> 人工序列
<220>
<223> B7R1[G25-P141] Fc5 Barbell (otPA前导序列)
<400> 16
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg
20 25 30
Phe Arg Arg Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys
35 40 45
Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln
50 55 60
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys
65 70 75 80
Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val
85 90 95
Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn
100 105 110
Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
115 120 125
Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu
130 135 140
His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp Lys Thr
145 150 155 160
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser
165 170 175
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
180 185 190
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
195 200 205
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
210 215 220
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
225 230 235 240
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
245 250 255
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr
260 265 270
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
275 280 285
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
290 295 300
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
305 310 315 320
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
325 330 335
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
340 345 350
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
355 360 365
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
Gly Gly Gly Ser Gly Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala
385 390 395 400
Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr
405 410 415
Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala
420 425 430
Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp
435 440 445
Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr
450 455 460
Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp
465 470 475 480
Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val
485 490 495
Ala Glu His Gly Ala Arg Phe Gln Ile Pro
500 505
<210> 17
<211> 1479
<212> DNA
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5 Barbell (EMIL前导序列)
<400> 17
atgcggcgcc gcggatggag ctggatcttt ctctttcttc tgtcaggaac tgcaggtgtc 60
ctctctggca caatagaaac aacggggaac atttctgcag agaaaggtgg ctctatcatc 120
ttacaatgtc acctctcctc caccacggca caagtgaccc aggtcaactg ggagcagcag 180
gaccagcttc tggccattta taatgctgac ttggggtggc acatctcccc atcgttcaag 240
gatcgagtgg ccccaggtcc cggcctgggc ctcaccctcc agtcgctgac cgtgaacgat 300
acaggggagt acttctgcat ctatcacacc taccctgatg ggacgtacac tgggagaatc 360
ttcctggagg tcctagaaag ctcagtggct gagcacggtg ccaggttcca gattccagag 420
cccaaatctt cagacaaaac tcacacatgc ccaccgtgcc cagcacctga agccgagggg 480
gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 540
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 600
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 660
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 720
aaggagtaca agtgcaaggt ctccaacaaa gccctcccat cctccatcga gaaaaccatc 780
tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccgggat 840
gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 900
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 960
gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg1020
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac1080
acgcagaaga gcctctccct gtctccgggt aaaggtggtg ggtccggagg tacaattgaa1140
acgacaggca atatatctgc cgagaagggt ggctcgatca tattacaatg ccacctctca1200
tccaccactg cacaagtaac ccaggtcaat tgggaacagc aggatcagct gctggccatc1260
tataacgctg acttgggatg gcacatttcc ccatcgttca aagatcgagt cgccccaggc1320
cccggcctcg gcctcacact ccagtctctg accgtcaacg atacaggcga gtacttttgc1380
atatatcaca cttaccctga cgggacctac actggtagaa tcttcctaga ggtcctagag1440
agctcagtcg ctgagcatgg ggctagattc cagatccca 1479
<210> 18
<211> 493
<212> PRT
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5 Barbell (EMIL前导序列)
<400> 18
Met Arg Arg Arg Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly
1 5 10 15
Thr Ala Gly Val Leu Ser Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser
20 25 30
Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr
35 40 45
Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu
50 55 60
Ala Ile Tyr Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
65 70 75 80
Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu
85 90 95
Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro
100 105 110
Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser
115 120 125
Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser
130 135 140
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly
145 150 155 160
Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
165 170 175
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
180 185 190
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
195 200 205
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
210 215 220
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
225 230 235 240
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile
245 250 255
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
260 265 270
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
275 280 285
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
290 295 300
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
305 310 315 320
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
325 330 335
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
340 345 350
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
355 360 365
Pro Gly Lys Gly Gly Gly Ser Gly Gly Thr Ile Glu Thr Thr Gly Asn
370 375 380
Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser
385 390 395 400
Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
405 410 415
Leu Leu Ala Ile Tyr Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser
420 425 430
Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln
435 440 445
Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr
450 455 460
Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu
465 470 475 480
Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro
485 490
<210> 19
<211> 1524
<212> DNA
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5 Tandem (EMIL前导序列)
<400> 19
atgcggcgcc gcggatggag ctggatcttt ctctttcttc tgtcaggaac tgcaggtgtc 60
ctctctggca caatagaaac aacggggaac atttctgcag agaaaggtgg ctctatcatc 120
ttacaatgtc acctctcctc caccacggca caagtgaccc aggtcaactg ggagcagcag 180
gaccagcttc tggccattta taatgctgac ttggggtggc acatctcccc atcgttcaag 240
gatcgagtgg ccccaggtcc cggcctgggc ctcaccctcc agtcgctgac cgtgaacgat 300
acaggggagt acttctgcat ctatcacacc taccctgatg ggacgtacac tgggagaatc 360
ttcctggagg tcctagaaag ctcagtggct gagcacggtg ccaggttcca gattccaggt 420
ggtggtggtt ctggaggagg aggatcaggc gggggtggat ccggaggtgg tggttctggt 480
acaattgaaa cgacaggcaa tatatctgcc gagaagggtg gctcgatcat attacaatgc 540
cacctctcat ccaccactgc acaagtaacc caggtcaatt gggaacagca ggatcagctg 600
ctggccatct ataacgctga cttgggatgg cacatttccc catcgttcaa agatcgagtc 660
gccccaggcc ccggcctcgg cctcacactc cagtctctga ccgtcaacga tacaggcgag 720
tacttttgca tatatcacac ttaccctgac gggacctaca ctggtagaat cttcctagag 780
gtcctagaga gctcagtcgc tgagcatggg gctagattcc agatcccaga gcccaaatct 840
tcagacaaaa ctcacacatg cccaccgtgc ccagcacctg aagccgaggg ggcaccgtca 900
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 960
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg1020
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg1080
taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac1140
aagtgcaagg tctccaacaa agccctccca tcctccatcg agaaaaccat ctccaaagcc1200
aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccggga tgagctgacc1260
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg1320
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1380
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag1440
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag1500
agcctctccc tgtctccggg taaa 1524
<210> 20
<211> 508
<212> PRT
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5 Tandem (EMIL前导序列)
<400> 20
Met Arg Arg Arg Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly
1 5 10 15
Thr Ala Gly Val Leu Ser Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser
20 25 30
Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr
35 40 45
Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu
50 55 60
Ala Ile Tyr Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys
65 70 75 80
Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu
85 90 95
Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro
100 105 110
Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser
115 120 125
Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile
165 170 175
Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln Val
180 185 190
Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Tyr Asn Ala Asp Leu
195 200 205
Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val Ala Pro Gly Pro
210 215 220
Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn Asp Thr Gly Glu
225 230 235 240
Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr Tyr Thr Gly Arg
245 250 255
Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu His Gly Ala Arg
260 265 270
Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
275 280 285
Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe
290 295 300
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
305 310 315 320
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
325 330 335
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
340 345 350
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
355 360 365
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
370 375 380
Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
385 390 395 400
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
405 410 415
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
420 425 430
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
435 440 445
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
450 455 460
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
465 470 475 480
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
485 490 495
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
500 505
<210> 21
<211> 1254
<212> DNA
<213> 智人
<400> 21
atggcccgag ccatggccgc cgcgtggccg ctgctgctgg tggcgctact ggtgctgtcc 60
tggccacccc caggaaccgg ggacgtcgtc gtgcaggcgc ccacccaggt gcccggcttc 120
ttgggcgact ccgtgacgct gccctgctac ctacaggtgc ccaacatgga ggtgacgcat 180
gtgtcacagc tgacttgggc gcggcatggt gaatctggca gcatggccgt cttccaccaa 240
acgcagggcc ccagctattc ggagtccaaa cggctggaat tcgtggcagc cagactgggc 300
gcggagctgc ggaatgcctc gctgaggatg ttcgggttgc gcgtagagga tgaaggcaac 360
tacacctgcc tgttcgtcac gttcccgcag ggcagcagga gcgtggatat ctggctccga 420
gtgcttgcca agccccagaa cacagctgag gttcagaagg tccagctcac tggagagcca 480
gtgcccatgg cccgctgcgt ctccacaggg ggtcgcccgc cagcccaaat cacctggcac 540
tcagacctgg gcgggatgcc caatacgagc caggtgccag ggttcctgtc tggcacagtc 600
actgtcacca gcctctggat attggtgccc tcaagccagg tggacggcaa gaatgtgacc 660
tgcaaggtgg agcacgagag ctttgagaag cctcagctgc tgactgtgaa cctcaccgtg 720
tactaccccc cagaggtatc catctctggc tatgataaca actggtacct tggccagaat 780
gaggccaccc tgacctgcga tgctcgcagc aacccagagc ccacaggcta taattggagc 840
acgaccatgg gtcccctgcc accctttgct gtggcccagg gcgcccagct cctgatccgt 900
cctgtggaca aaccaatcaa cacaacttta atctgcaacg tcaccaatgc cctaggagct 960
cgccaggcag aactgaccgt ccaggtcaaa gagggacctc ccagtgagca ctcaggcatg1020
tcccgtaacg ccatcatctt cctggttctg ggaatcctgg tttttctgat cctgctgggg1080
atcgggattt atttctattg gtccaaatgt tcccgtgagg tcctttggca ctgtcatctg1140
tgtccctcga gtacagagca tgccagcgcc tcagctaatg ggcatgtctc ctattcagct1200
gtgagcagag agaacagctc ttcccaggat ccacagacag agggcacaag gtga 1254
<210> 22
<211> 417
<212> PRT
<213> 智人
<400> 22
Met Ala Arg Ala Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu
1 5 10 15
Leu Val Leu Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln
20 25 30
Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro
35 40 45
Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu
50 55 60
Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln
65 70 75 80
Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala
85 90 95
Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly
100 105 110
Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe
115 120 125
Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys
130 135 140
Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro
145 150 155 160
Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln
165 170 175
Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val
180 185 190
Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu
195 200 205
Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu
210 215 220
His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val
225 230 235 240
Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr
245 250 255
Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro
260 265 270
Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro
275 280 285
Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys
290 295 300
Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala
305 310 315 320
Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu
325 330 335
His Ser Gly Met Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile
340 345 350
Leu Val Phe Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp Ser
355 360 365
Lys Cys Ser Arg Glu Val Leu Trp His Cys His Leu Cys Pro Ser Ser
370 375 380
Thr Glu His Ala Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala
385 390 395 400
Val Ser Arg Glu Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr
405 410 415
Arg
<210> 23
<211> 1227
<212> DNA
<213> 小家鼠
<400> 23
atggctcaac tcgcccgagc cacccgctcc ccgctgtcat ggctgctgct gctgttctgc 60
tatgcactcc ggaaagcggg tggggatata cgtgtgctgg tgccctacaa ttcgacaggc 120
gtcttgggag ggtcgaccac cttgcactgt agtctgactt ctaatgagaa tgtgactatc 180
actcaaataa cctggatgaa gaaggattca ggtggatccc acgctcttgt ggctgtcttc 240
caccccaaga aggggcccaa catcaaagag ccagagaggg tgaaattctt ggctgcccaa 300
caggatctga ggaacgcatc tctggccatc tcgaacttaa gtgtagaaga cgaaggcatc 360
tatgaatgtc agattgccac attccccaga ggcagtagaa gcaccaatgc ctggctgaag 420
gtgcaagccc gacctaagaa cactgcagag gccctggagc cctctcccac cttgatactg 480
caggatgtgg ctaaatgcat ctctgccaat ggtcaccctc ctggacgaat ctcttggccc 540
tcgaatgtga atggaagtca ccgtgaaatg aaggaaccag ggtcccagcc gggcaccacc 600
acagttacca gctacctctc catggtacct tctcgccagg cagacggcaa gaacatcacc 660
tgcacggtgg agcatgaaag cttacaggag ctggaccagc tgctggtgac cctttcccaa 720
ccctatccac ctgaaaacgt gtccatctct ggctatgacg gcaactggta tgttggcctc 780
actaacttga ccctgacctg tgaagctcac agcaaaccag cgcctgacat ggctggatat 840
aactggagca cgaacacggg tgactttccc aactctgtta agcgccaggg caatatgctt 900
ctaatctcca ccgtagagga tggtctcaat aacacggtca ttgtgtgcga agtcaccaat 960
gccctagggt ctgggcaggg ccaagtgcac atcattgtta aagagaaacc tgagaatatg1020
cagcaaaata caagattaca cctaggctac atctttctta tcgtctttgt cctcgctgta1080
gtcatcatca tcgcagcact atacactata cgaagatgca ggcatggtcg tgctctgcag1140
tccaatccct cagagaggga gaacgtccag tattcatctg tgaacggcga ctgtagactg1200
aacatggagc caaacagcac aaggtga 1227
<210> 24
<211> 408
<212> PRT
<213> 小家鼠
<400> 24
Met Ala Gln Leu Ala Arg Ala Thr Arg Ser Pro Leu Ser Trp Leu Leu
1 5 10 15
Leu Leu Phe Cys Tyr Ala Leu Arg Lys Ala Gly Gly Asp Ile Arg Val
20 25 30
Leu Val Pro Tyr Asn Ser Thr Gly Val Leu Gly Gly Ser Thr Thr Leu
35 40 45
His Cys Ser Leu Thr Ser Asn Glu Asn Val Thr Ile Thr Gln Ile Thr
50 55 60
Trp Met Lys Lys Asp Ser Gly Gly Ser His Ala Leu Val Ala Val Phe
65 70 75 80
His Pro Lys Lys Gly Pro Asn Ile Lys Glu Pro Glu Arg Val Lys Phe
85 90 95
Leu Ala Ala Gln Gln Asp Leu Arg Asn Ala Ser Leu Ala Ile Ser Asn
100 105 110
Leu Ser Val Glu Asp Glu Gly Ile Tyr Glu Cys Gln Ile Ala Thr Phe
115 120 125
Pro Arg Gly Ser Arg Ser Thr Asn Ala Trp Leu Lys Val Gln Ala Arg
130 135 140
Pro Lys Asn Thr Ala Glu Ala Leu Glu Pro Ser Pro Thr Leu Ile Leu
145 150 155 160
Gln Asp Val Ala Lys Cys Ile Ser Ala Asn Gly His Pro Pro Gly Arg
165 170 175
Ile Ser Trp Pro Ser Asn Val Asn Gly Ser His Arg Glu Met Lys Glu
180 185 190
Pro Gly Ser Gln Pro Gly Thr Thr Thr Val Thr Ser Tyr Leu Ser Met
195 200 205
Val Pro Ser Arg Gln Ala Asp Gly Lys Asn Ile Thr Cys Thr Val Glu
210 215 220
His Glu Ser Leu Gln Glu Leu Asp Gln Leu Leu Val Thr Leu Ser Gln
225 230 235 240
Pro Tyr Pro Pro Glu Asn Val Ser Ile Ser Gly Tyr Asp Gly Asn Trp
245 250 255
Tyr Val Gly Leu Thr Asn Leu Thr Leu Thr Cys Glu Ala His Ser Lys
260 265 270
Pro Ala Pro Asp Met Ala Gly Tyr Asn Trp Ser Thr Asn Thr Gly Asp
275 280 285
Phe Pro Asn Ser Val Lys Arg Gln Gly Asn Met Leu Leu Ile Ser Thr
290 295 300
Val Glu Asp Gly Leu Asn Asn Thr Val Ile Val Cys Glu Val Thr Asn
305 310 315 320
Ala Leu Gly Ser Gly Gln Gly Gln Val His Ile Ile Val Lys Glu Lys
325 330 335
Pro Glu Asn Met Gln Gln Asn Thr Arg Leu His Leu Gly Tyr Ile Phe
340 345 350
Leu Ile Val Phe Val Leu Ala Val Val Ile Ile Ile Ala Ala Leu Tyr
355 360 365
Thr Ile Arg Arg Cys Arg His Gly Arg Ala Leu Gln Ser Asn Pro Ser
370 375 380
Glu Arg Glu Asn Val Gln Tyr Ser Ser Val Asn Gly Asp Cys Arg Leu
385 390 395 400
Asn Met Glu Pro Asn Ser Thr Arg
405
<210> 25
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 多肽接头
<400> 25
Gly Gly Gly Ser Gly
1 5
<210> 26
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 多肽接头
<220>
<221> misc_feature
<222> (16)..(19)
<223> Xaa是Gly或不存在
<220>
<221> misc_feature
<222> (20)..(20)
<223> Xaa是Ser或不存在
<220>
<221> misc_feature
<222> (21)..(24)
<223> Xaa是Gly或不存在
<220>
<221> misc_feature
<222> (25)..(25)
<223> Xaa是Ser或不存在
<400> 26
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25
<210> 27
<211> 330
<212> PRT
<213> 智人
<400> 27
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 28
<211> 232
<212> PRT
<213> 智人
<400> 28
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 29
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> Fc-488
<400> 29
Glu Pro Arg Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 30
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> Fc4
<400> 30
Glu Pro Arg Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 31
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> Fc5
<400> 31
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 32
<211> 231
<212> PRT
<213> 人工序列
<220>
<223> Fc6
<400> 32
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly
225 230
<210> 33
<211> 232
<212> PRT
<213> 人工序列
<220>
<223> Fc7
<400> 33
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Glu Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 34
<211> 346
<212> PRT
<213> 人工序列
<220>
<223> mB7R1-mFc2
<400> 34
Gly Thr Ile Asp Thr Lys Arg Asn Ile Ser Ala Glu Glu Gly Gly Ser
1 5 10 15
Val Ile Leu Gln Cys His Phe Ser Ser Asp Thr Ala Glu Val Thr Gln
20 25 30
Val Asp Trp Lys Gln Gln Asp Gln Leu Leu Ala Ile Tyr Ser Val Asp
35 40 45
Leu Gly Trp His Val Ala Ser Val Phe Ser Asp Arg Val Val Pro Gly
50 55 60
Pro Ser Leu Gly Leu Thr Phe Gln Ser Leu Thr Met Asn Asp Thr Gly
65 70 75 80
Glu Tyr Phe Cys Thr Tyr His Thr Tyr Pro Gly Gly Ile Tyr Lys Gly
85 90 95
Arg Ile Phe Leu Lys Val Gln Glu Ser Ser Val Ala Gln Phe Gln Thr
100 105 110
Ala Glu Pro Arg Ser Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
115 120 125
Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
130 135 140
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
145 150 155 160
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
165 170 175
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
180 185 190
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
195 200 205
His Gln Asp Trp Met Ser Gly Lys Ala Phe Ala Cys Ala Val Asn Asn
210 215 220
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
225 230 235 240
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
245 250 255
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
260 265 270
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
275 280 285
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
290 295 300
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
305 310 315 320
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
325 330 335
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
340 345
<210> 35
<211> 168
<212> PRT
<213> 人工序列
<220>
<223> mB7R1-VASP
<400> 35
Gly Thr Ile Asp Thr Lys Arg Asn Ile Ser Ala Glu Glu Gly Gly Ser
1 5 10 15
Val Ile Leu Gln Cys His Phe Ser Ser Asp Thr Ala Glu Val Thr Gln
20 25 30
Val Asp Trp Lys Gln Gln Asp Gln Leu Leu Ala Ile Tyr Ser Val Asp
35 40 45
Leu Gly Trp His Val Ala Ser Val Phe Ser Asp Arg Val Val Pro Gly
50 55 60
Pro Ser Leu Gly Leu Thr Phe Gln Ser Leu Thr Met Asn Asp Thr Gly
65 70 75 80
Glu Tyr Phe Cys Thr Tyr His Thr Tyr Pro Gly Gly Ile Tyr Lys Gly
85 90 95
Arg Ile Phe Leu Lys Val Gln Glu Ser Ser Val Ala Gln Phe Gln Thr
100 105 110
Ala Glu Pro Arg Ser Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Gln
115 120 125
Arg Val Lys Gln Glu Leu Leu Glu Glu Val Lys Lys Glu Leu Gln Lys
130 135 140
Val Lys Glu Glu Ile Ile Glu Ala Phe Val Gln Glu Leu Arg Gly Ser
145 150 155 160
Gly Gly His His His His His His
165
<210> 36
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60639)
<400> 36
caggtgtcca gggaattcat ataggccggc caccatgcat ggctggctgc tcctg 55
<210> 37
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60643)
<400> 37
cggccgcatt cctgaggttt ttatccggac ccaccacctt tacccggagt ccgggagaag 60
c 61
<210> 38
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60645)
<400> 38
caaccccaga gctgttttaa ggcgcgcctc tagaaacggc cgcattcctg aggttttta 59
<210> 39
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60642)
<400> 39
gtggtgggtc cggaggcacg atagatacaa agag 34
<210> 40
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60641)
<400> 40
gcgcctctag aaacggccgc attcctgagg tttttaggca gtctggaac 49
<210> 41
<211> 64
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc53051)
<400> 41
caggtgtcca gggaattcat ataggccggc caccatgcgc tggtgtctcc tcctgatctg 60
ggcc 64
<210> 42
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60385)
<400> 42
gagttttgtc tgaagatttg ggctctggaa tctggaacct ggcaccg 47
<210> 43
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60386
<400> 43
caggttccag attccagagc ccaaatcttc agacaaaact cacac 45
<210> 44
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc59433)
<400> 44
aaagatctca ttcctgaggt ttttatccgg acccaccacc tttacccgga gacagggaga 60
g 61
<210> 45
<211> 62
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc59434)
<400> 45
caaccccaga gctgttttaa ggcgcgcctc tagattaaaa gatctcattc ctgaggtttt 60
ta 62
<210> 46
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60640)
<400> 46
gatcctgagc ccgaaccgcc ggcagtctgg aactgagcca c 41
<210> 47
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60644)
<400> 47
gcctccggac ccaccaccag aaccaccgga acctgaacct cccgatcctg agcccgaacc 60
gcc 63
<210> 48
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60642)
<400> 48
gtggtgggtc cggaggcacg atagatacaa agag 34
<210> 49
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc28844)
<400> 49
gacggatggt ccaccctcga ggttaggtgc tgg 33
<210> 50
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc62529)
<400> 50
cgatcctgag cccgaaccgc ctggaatctg gaacctggca ccgtg 45
<210> 51
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc62530)
<400> 51
accagatccg gacccaccac cagaaccacc ggaacctgaa cctcccgatc ctgagcccga 60
accgcc 66
<210> 52
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc62531)
<400> 52
ctggtggtgg gtccggaatg atgacaggca caatagaaac aac 43
<210> 53
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc62532)
<400> 53
accagaaaga tctcattcct gaggttttta tttacccgga gacagggaga ggct 54
<210> 54
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64230)
<400> 54
caggaaatcc atgccgagtt gagacgcttc cgtagaggca caatagaaac aacggggaac 60
<210> 55
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64219)
<400> 55
cttgaaggat ggggagatgt gccaccccaa gtcagcatta caaatggcca gaagctggtc 60
ctgctg 66
<210> 56
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64215)
<400> 56
aatgctgact tggggtggca catctcccca tccttcaagg atcgagtggc cccaggtccc 60
<210> 57
<211> 60
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64216)
<400> 57
tccggaccca ccacctttac ccggagacag ggagaggctc ttctgcgtgt agtggttgtg 60
<210> 58
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64228)
<400> 58
aagagcctct ccctgtctcc gggtaaaggt ggtgggtccg gaggtacaat tgaaacgaca 60
ggcaat 66
<210> 59
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64220)
<400> 59
gtggcattgt aatatgatcg agccaccctt ctcggcagat atattgcctg tcgtttcaat 60
tgt 63
<210> 60
<211> 73
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64224)
<400> 60
gatcatatta caatgccacc tctcatccac cactgcacaa gtaacccagg tcaattggga 60
acagcaggat cag 73
<210> 61
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64231)
<400> 61
cgatggggaa atgtgccatc ccaagtcagc gttacagatg gccagcagct gatcctgctg 60
ttccca 66
<210> 62
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64225)
<400> 62
tggcacattt ccccatcgtt caaagatcga gtcgccccag gccccggcct cggcctcaca 60
ctc 63
<210> 63
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64221)
<400> 63
gtgatatatg caaaagtact cgcctgtatc gttgacggtc agagactgga gtgtgaggcc 60
gaggcc 66
<210> 64
<211> 68
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64226)
<400> 64
gtacttttgc atatatcaca cttaccctga cgggacctac actggtagaa tcttcctaga 60
ggtcctag 68
<210> 65
<211> 65
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64222)
<400> 65
ttatgggatc tggaatctag ccccatgctc agcgactgag ctctctagga cctctaggaa 60
gattc 65
<210> 66
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64223)
<400> 66
taaggcgcgc ctctagatta aaagatctca ttcctgaggt ttttatggga tctggaatct 60
agccccatg 69
<210> 67
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64258)
<400> 67
catggggcta gattccagat cccataaaaa cctcaggaat gagatctttt aatctagagg 60
cgcgcctta 69
<210> 68
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64218)
<400> 68
cttgaaggat ggggagatgt gccaccccaa gtcagcatta taaatggcca gaagctggtc 60
ctgctg 66
<210> 69
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc64227)
<400> 69
cgatggggaa atgtgccatc ccaagtcagc gttatagatg gccagcagct gatcctgctg 60
ttccca 66
<210> 70
<211> 82
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65030)
<400> 70
tctccacagg tgtccaggga attcatatag gccggccacc atgcggcgcc gcggatggag 60
ctggatcttt ctctttcttc tg 82
<210> 71
<211> 72
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65029)
<400> 71
agctggatct ttctctttct tctgtcagga actgcaggtg tcctctctgg cacaatagaa 60
acaacgggga ac 72
<210> 72
<211> 82
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65050)
<400> 72
agaaccacca cctccggatc cacccccgcc tgatcctcct cctccagaac caccaccacc 60
tggaatctgg aacctggcac cg 82
<210> 73
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65051)
<400> 73
ggaggaggag gatcaggcgg gggtggatcc ggaggtggtg gttctggtac aattgaaacg 60
acaggcaat 69
<210> 74
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65052)
<400> 74
cggtgggcat gtgtgagttt tgtctgaaga tttgggctct gggatctgga atctagcccc 60
a 61
<210> 75
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65053)
<400> 75
gagcccaaat cttcagacaa aactcacaca tgcccaccgt gcccagcacc tgaagcc 57
<210> 76
<211> 85
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc65054)
<400> 76
caaccccaga gctgttttaa ggcgcgcctc tagattaaaa gatctcattc ctgaggtttt 60
tatttacccg gagacaggga gaggc 85
<210> 77
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc59435)
<400> 77
ggtaaaggtg gtgggtccgg aatgatgaca ggcacaatag aaac 44
<210> 78
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸引物(zc60392)
<400> 78
ctagattaaa agatctcatt cctgaggttt ttatggaatc tggaacctgg caccg 55
<210> 79
<211> 352
<212> PRT
<213> 人工序列
<220>
<223> B7R1-Fc2
<400> 79
Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys
1 5 10 15
Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln
20 25 30
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys
35 40 45
Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val
50 55 60
Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn
65 70 75 80
Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Ala
85 90 95
Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu
100 105 110
His Gly Ala Arg Phe Gln Ile Glu Pro Arg Ser Pro Thr Ile Lys Pro
115 120 125
Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser
130 135 140
Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu
145 150 155 160
Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro
165 170 175
Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala
180 185 190
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val
195 200 205
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Ala Phe
210 215 220
Ala Cys Ala Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
225 230 235 240
Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu
245 250 255
Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys
260 265 270
Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn
275 280 285
Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp
290 295 300
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys
305 310 315 320
Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly
325 330 335
Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
340 345 350
<210> 80
<211> 349
<212> PRT
<213> 人工序列
<220>
<223> B7R1[G25-P141] C69Y Fc5
<400> 80
Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys Gly Gly Ser
1 5 10 15
Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln
20 25 30
Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Tyr Asn Ala Asp
35 40 45
Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val Ala Pro Gly
50 55 60
Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn Asp Thr Gly
65 70 75 80
Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr Tyr Thr Gly
85 90 95
Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu His Gly Ala
100 105 110
Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
115 120 125
Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu
130 135 140
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
145 150 155 160
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
165 170 175
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
180 185 190
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
210 215 220
Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 81
<211> 173
<212> PRT
<213> 人工序列
<220>
<223> B7R1-VASP
<400> 81
Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys
1 5 10 15
Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln
20 25 30
Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys
35 40 45
Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val
50 55 60
Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn
65 70 75 80
Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr
85 90 95
Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu
100 105 110
His Gly Ala Arg Phe Gln Ile Pro Arg Ser Gly Ser Gly Gly Ser Gly
115 120 125
Gly Ser Asp Leu Gln Arg Val Lys Gln Glu Leu Leu Glu Glu Val Lys
130 135 140
Lys Glu Leu Gln Lys Val Lys Glu Glu Ile Ile Glu Ala Phe Val Gln
145 150 155 160
Glu Leu Arg Gly Ser Gly Gly His His His His His His
165 170
Claims (15)
1.可溶性多肽融合体,其从氨基端至羧基端由P1-L1-D-L2-P2组成,其中:
P1是SEQ ID NO:2的氨基酸残基25-141或SEQ ID NO:18的氨基酸残基23-139所示的第一种多肽;
L1是第一种多肽接头;
D是二聚化结构域;
L2是第二种多肽接头;
P2是SEQ ID NO:2的氨基酸残基25-141或SEQ ID NO:18的氨基酸残基23-139所示的第二种多肽;
其中所述多肽融合体能够特异性地结合CD155的细胞外结构域,即SEQ ID NO:22的氨基酸残基28-343。
2.可溶性多肽融合体,其从氨基端至羧基端由P2-L2-P1-L1-D组成,其中:
P1是SEQ ID NO:2的氨基酸残基25-141或SEQ ID NO:18的氨基酸残基23-139所示的第一种多肽;
L1是第一种多肽接头;
D是二聚化结构域;
L2是第二种多肽接头;
P2是SEQ ID NO:2的氨基酸残基25-141或SEQ ID NO:18的氨基酸残基23-139所示的第二种多肽;
其中所述多肽融合体能够特异性地结合CD155的细胞外结构域,即SEQ ID NO:22的氨基酸残基28-343。
3.权利要求1或2的多肽融合体,其中D是免疫球蛋白重链恒定区。
4.权利要求1或2的多肽融合体,其中L1由15-32个氨基酸残基组成,其中所述残基中的1-8个是半胱氨酸残基。
5.权利要求1或2的多肽融合体,其中L2包含多个甘氨酸残基,且任选地包含至少一个丝氨酸残基。
6.权利要求1或2的多肽融合体,其中L2由在SEQ ID NO:25中所示的氨基酸序列组成。
7.权利要求1的多肽融合体,其中所述多肽融合体由在SEQ ID NO:18的残基23-493中所示的氨基酸序列组成。
8.权利要求2的多肽融合体,其中所述多肽融合体由在SEQ ID NO:20的残基23-508或23-507中所示的氨基酸序列组成。
9.多核苷酸,其编码权利要求1-8中任一项的多肽融合体。
10.表达载体,其包含下述可操作地连接的元件:
转录启动子;
DNA区段,其编码权利要求1-8中任一项的多肽融合体;和
转录终止子。
11.培养的细胞,其中已经引入权利要求10的表达载体,其中所述细胞表达所述DNA区段。
12.制备多肽融合体的方法,所述方法包括:
培养细胞,所述细胞中已经引入权利要求10的表达载体,其中所述细胞表达所述DNA区段,并生产编码的多肽融合体;和
回收所述多肽融合体。
13.组合物,其包含:
权利要求1-8中的任一项的多肽融合体;和
药学上可接受的载体。
14.权利要求1-8中的任一项的多肽融合体在制备药物中的用途,所述药物用于在受试者中治疗T-细胞介导的免疫障碍。
15.权利要求14的用途,其中所述自身免疫病选自类风湿性关节炎和多发性硬化。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US35287310P | 2010-06-09 | 2010-06-09 | |
US61/352873 | 2010-06-09 | ||
PCT/US2011/039422 WO2011156356A1 (en) | 2010-06-09 | 2011-06-07 | Dimeric vstm3 fusion proteins and related compositions and methods |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103168048A CN103168048A (zh) | 2013-06-19 |
CN103168048B true CN103168048B (zh) | 2017-02-15 |
Family
ID=44281053
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201180039006.4A Active CN103168048B (zh) | 2010-06-09 | 2011-06-07 | 二聚vstm3融合蛋白和相关的组合物和方法 |
Country Status (9)
Country | Link |
---|---|
US (1) | US8822642B2 (zh) |
EP (1) | EP2580238A1 (zh) |
JP (1) | JP2013531982A (zh) |
CN (1) | CN103168048B (zh) |
BR (1) | BR112012031329A2 (zh) |
CA (1) | CA2802017A1 (zh) |
EA (1) | EA022932B1 (zh) |
MX (1) | MX2012013899A (zh) |
WO (1) | WO2011156356A1 (zh) |
Families Citing this family (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1539228B1 (en) | 2002-09-11 | 2010-12-29 | Genentech, Inc. | Novel composition and methods for the treatment of immune related diseases |
US20090258013A1 (en) | 2008-04-09 | 2009-10-15 | Genentech, Inc. | Novel compositions and methods for the treatment of immune related diseases |
DK2665486T3 (da) * | 2011-01-18 | 2020-03-09 | Bioniz Llc | Sammensætninger til modulering af gamma-c-cytokin-aktivitet |
CN103965362B (zh) * | 2013-02-06 | 2019-02-01 | 上海细胞治疗集团有限公司 | 一种可使t细胞趋向肿瘤部位的嵌合趋化因子受体 |
RS60826B1 (sr) | 2013-07-16 | 2020-10-30 | Hoffmann La Roche | Postupci lečenja kancera upotrebom antagonista vezivanja pd-1 ose i tigit inhibitora |
US9959384B2 (en) * | 2013-12-10 | 2018-05-01 | Bioniz, Llc | Methods of developing selective peptide antagonists |
JP6920194B2 (ja) | 2014-08-19 | 2021-08-18 | メルク・シャープ・アンド・ドーム・コーポレーションMerck Sharp & Dohme Corp. | 抗tigit抗体 |
US11566082B2 (en) | 2014-11-17 | 2023-01-31 | Cytiva Bioprocess R&D Ab | Mutated immunoglobulin-binding polypeptides |
MA40662B1 (fr) | 2014-12-23 | 2020-12-31 | Bristol Myers Squibb Co | Anticorps contre tigit |
AU2016307845B2 (en) | 2015-08-14 | 2020-10-15 | Merck Sharp & Dohme Llc | Anti-TIGIT antibodies |
RU2732591C2 (ru) | 2015-09-25 | 2020-09-21 | Дженентек, Инк. | Анти-tigit антитела и способы применения |
CN108368176B (zh) | 2015-10-01 | 2022-06-07 | 波滕扎治疗公司 | 抗tigit抗原结合蛋白及其使用方法 |
EP3359556B1 (en) | 2015-10-09 | 2021-05-26 | Bioniz, LLC | Modulating gamma - c -cytokine activity |
BR112018012844A2 (pt) * | 2015-12-23 | 2018-12-04 | Greenovation Biotech Gmbh | polipeptídeos para inibir a ativação de complemento |
KR102588359B1 (ko) | 2016-03-04 | 2023-10-13 | 제이엔 바이오사이언시즈 엘엘씨 | Tigit에 대한 항체 |
CN109072226B (zh) | 2016-04-20 | 2021-10-15 | 国立大学法人筑波大学 | 调节性t细胞的激活剂及其应用 |
JP7106187B2 (ja) | 2016-05-11 | 2022-07-26 | サイティバ・バイオプロセス・アールアンドディ・アクチボラグ | 分離マトリックスを保存する方法 |
US10654887B2 (en) | 2016-05-11 | 2020-05-19 | Ge Healthcare Bio-Process R&D Ab | Separation matrix |
US10513537B2 (en) | 2016-05-11 | 2019-12-24 | Ge Healthcare Bioprocess R&D Ab | Separation matrix |
CN109311948B (zh) | 2016-05-11 | 2022-09-16 | 思拓凡生物工艺研发有限公司 | 清洁和/或消毒分离基质的方法 |
US10730908B2 (en) | 2016-05-11 | 2020-08-04 | Ge Healthcare Bioprocess R&D Ab | Separation method |
US10703774B2 (en) | 2016-09-30 | 2020-07-07 | Ge Healthcare Bioprocess R&D Ab | Separation method |
US10889615B2 (en) | 2016-05-11 | 2021-01-12 | Cytiva Bioprocess R&D Ab | Mutated immunoglobulin-binding polypeptides |
JOP20190203A1 (ar) | 2017-03-30 | 2019-09-03 | Potenza Therapeutics Inc | بروتينات رابطة لمولد ضد مضادة لـ tigit وطرق استخدامها |
CN110997720A (zh) | 2017-07-27 | 2020-04-10 | Iteos治疗公司 | 抗tigit抗体 |
CN108558997B (zh) * | 2017-10-20 | 2021-10-08 | 中国人民解放军第四军医大学 | 一种重组融合蛋白TIGIT-Fc及其抗移植排斥反应的应用 |
WO2020018935A2 (en) * | 2018-07-19 | 2020-01-23 | University Of Washington | De novo design of protein switches |
KR20210057053A (ko) | 2018-08-23 | 2021-05-20 | 씨젠 인크. | 항-tigit 항체 |
US11312963B2 (en) | 2018-10-18 | 2022-04-26 | Synerk Inc. | Compositions and methods for inhibiting TIGIT gene expression |
CN110669139A (zh) * | 2019-09-18 | 2020-01-10 | 沣潮医药科技(上海)有限公司 | 二聚体免疫粘附素、药物组合物和用途 |
CN111018999B (zh) * | 2019-12-05 | 2022-07-12 | 沣潮医药科技(上海)有限公司 | 二聚体免疫融合蛋白、药物组合物和用途 |
IL298111A (en) | 2020-06-02 | 2023-01-01 | Arcus Biosciences Inc | Antibodies to tigit |
CN112190689B (zh) * | 2020-08-25 | 2022-12-09 | 沣潮医药科技(上海)有限公司 | Tigit免疫粘附素在调控肿瘤免疫及调节血管生成产品中的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006124667A2 (en) * | 2005-05-12 | 2006-11-23 | Zymogenetics, Inc. | Compositions and methods for modulating immune responses |
WO2009046407A2 (en) * | 2007-10-04 | 2009-04-09 | Zymogenetics, Inc. | B7 FAMILY MEMBER zB7H6 AND RELATED COMPOSITIONS AND METHODS |
Family Cites Families (53)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU560472B2 (en) | 1981-08-25 | 1987-04-09 | Celltech Limited | Yeast expression vectors |
US4579821A (en) | 1981-11-23 | 1986-04-01 | University Patents, Inc. | Control of DNA sequence transcription |
US4656134A (en) | 1982-01-11 | 1987-04-07 | Board Of Trustees Of Leland Stanford Jr. University | Gene amplification in eukaryotic cells |
US4486533A (en) | 1982-09-02 | 1984-12-04 | St. Louis University | Filamentous fungi functional replicating extrachromosomal element |
US4599311A (en) | 1982-08-13 | 1986-07-08 | Kawasaki Glenn H | Glycolytic promotersfor regulated protein expression: protease inhibitor |
US4977092A (en) | 1985-06-26 | 1990-12-11 | Amgen | Expression of exogenous polypeptides and polypeptide products including hepatitis B surface antigen in yeast cells |
US4601978A (en) | 1982-11-24 | 1986-07-22 | The Regents Of The University Of California | Mammalian metallothionein promoter system |
US4713339A (en) | 1983-01-19 | 1987-12-15 | Genentech, Inc. | Polycistronic expression vector construction |
US4661454A (en) | 1983-02-28 | 1987-04-28 | Collaborative Research, Inc. | GAL1 yeast promoter linked to non galactokinase gene |
US5139936A (en) | 1983-02-28 | 1992-08-18 | Collaborative Research, Inc. | Use of the GAL1 yeast promoter |
US4870008A (en) | 1983-08-12 | 1989-09-26 | Chiron Corporation | Secretory expression in eukaryotes |
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 |
EP0184576B1 (en) | 1984-12-06 | 1990-06-27 | Fina Research S.A. | Promoters for the expression of foreign genes in yeast, plasmids comprising them, and uses thereof for the production of polypeptides |
US5208036A (en) | 1985-01-07 | 1993-05-04 | Syntex (U.S.A.) Inc. | N-(ω, (ω-1)-dialkyloxy)- and N-(ω, (ω-1)-dialkenyloxy)-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
GR860984B (en) | 1985-04-17 | 1986-08-18 | Zymogenetics Inc | Expression of factor vii and ix activities in mammalian cells |
US4882279A (en) | 1985-10-25 | 1989-11-21 | Phillips Petroleum Company | Site selective genomic modification of yeast of the genus pichia |
US4935349A (en) | 1986-01-17 | 1990-06-19 | Zymogenetics, Inc. | Expression of higher eucaryotic genes in aspergillus |
GB8611832D0 (en) | 1986-05-15 | 1986-06-25 | Holland I B | Polypeptide |
US5063154A (en) | 1987-06-24 | 1991-11-05 | Whitehead Institute For Biomedical Research | Pheromone - inducible yeast promoter |
ATE140963T1 (de) | 1988-01-22 | 1996-08-15 | Zymogenetics Inc | Verfahren zur herstellung von sekretierten rezeptoranalogen |
US5567584A (en) | 1988-01-22 | 1996-10-22 | Zymogenetics, Inc. | Methods of using biologically active dimerized polypeptide fusions to detect PDGF |
US4956288A (en) | 1988-04-22 | 1990-09-11 | Biogen, Inc. | Method for producing cells containing stably integrated foreign DNA at a high copy number, the cells produced by this method, and the use of these cells to produce the polypeptides coded for by the foreign DNA |
US5037743A (en) | 1988-08-05 | 1991-08-06 | Zymogenetics, Inc. | BAR1 secretion signal |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5162228A (en) | 1988-12-28 | 1992-11-10 | Takeda Chemical Industries, Ltd. | Gylceraldehyde-3-phosphate dehydrogenase gene and promoter |
US5399346A (en) | 1989-06-14 | 1995-03-21 | The United States Of America As Represented By The Department Of Health And Human Services | Gene therapy |
US5162222A (en) | 1989-07-07 | 1992-11-10 | Guarino Linda A | Use of baculovirus early promoters for expression of foreign genes in stably transformed insect cells or recombinant baculoviruses |
US5605690A (en) | 1989-09-05 | 1997-02-25 | Immunex Corporation | Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor |
US5279833A (en) | 1990-04-04 | 1994-01-18 | Yale University | Liposomal transfection of nucleic acids into animal cells |
US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
IL99552A0 (en) | 1990-09-28 | 1992-08-18 | Ixsys Inc | Compositions containing procaryotic cells,a kit for the preparation of vectors useful for the coexpression of two or more dna sequences and methods for the use thereof |
JP3202999B2 (ja) | 1991-01-31 | 2001-08-27 | 協和醗酵工業株式会社 | 肝移行性リポソーム製剤 |
PT503648E (pt) | 1991-03-12 | 2000-10-31 | Biogen Inc | Dominio do antigenio 3 associado a funcao linfocitica de ligacao as cd2 |
US5283185A (en) | 1991-08-28 | 1994-02-01 | University Of Tennessee Research Corporation | Method for delivering nucleic acids into cells |
US5298418A (en) | 1991-09-16 | 1994-03-29 | Boyce Thompson Institute For Plant Research, Inc. | Cell line isolated from larval midgut tissue of Trichoplusia ni |
CA2144651A1 (en) | 1992-09-14 | 1994-03-31 | Timothy J. Miller | Immortalized cells and uses therefor |
AU5670194A (en) | 1992-11-20 | 1994-06-22 | Enzon, Inc. | Linker for linked fusion polypeptides |
US5674703A (en) | 1992-12-02 | 1997-10-07 | Woo; Savio L. C. | Episomal vector systems and related methods |
WO1995005853A1 (en) | 1993-08-26 | 1995-03-02 | The Regents Of The University Of California | Method, compositions and devices for administration of naked polynucleotides which encode biologically active peptides |
US6117679A (en) | 1994-02-17 | 2000-09-12 | Maxygen, Inc. | Methods for generating polynucleotides having desired characteristics by iterative selection and recombination |
US5641655A (en) | 1994-11-30 | 1997-06-24 | Zymogenetics, Inc. | Methods for producing thrombopoietin polypeptides using a mammalian tissue plasminogen activator secretory peptide |
US5955349A (en) | 1996-08-26 | 1999-09-21 | Zymogenetics, Inc. | Compositions and methods for producing heterologous polypeptides in Pichia methanolica |
US5716808A (en) | 1995-11-09 | 1998-02-10 | Zymogenetics, Inc. | Genetic engineering of pichia methanolica |
CN1230997A (zh) | 1996-07-17 | 1999-10-06 | 津莫吉尼蒂克斯公司 | 甲醇毕赤酵母的转化 |
US5736383A (en) | 1996-08-26 | 1998-04-07 | Zymogenetics, Inc. | Preparation of Pichia methanolica auxotrophic mutants |
SI2116259T1 (sl) | 2001-05-24 | 2012-11-30 | Zymogenetics Inc | Taci imunoglobulin fuzijski proteini |
CA2488924A1 (en) | 2002-06-18 | 2003-12-24 | Zymogenetics, Inc. | Hybrid vector having a cytomegalovirus enhancer and myeloproliferative sarcoma virus promoter |
WO2007124283A2 (en) | 2006-04-13 | 2007-11-01 | Zymogenetics, Inc. | Tetramerizing polypeptides and methods of use |
WO2008013782A2 (en) * | 2006-07-24 | 2008-01-31 | Biogen Idec Ma Inc. | Methods for promoting myelination, neuronal survival and oligodendrocyte differentiation via administration of sp35 or trka antagonists |
CA2660732A1 (en) * | 2006-08-31 | 2008-03-06 | Biogen Idec Ma Inc. | Methods relating to peripheral administration of nogo receptor polypeptides |
BRPI0921837A2 (pt) * | 2008-11-25 | 2016-01-12 | Biogen Idec Inc | anticorpos isolados ou fragmentos de antígeno de ligação dos mesmos que podem se ligar especificamente a um polipeptídio dr6, métodos in vitro de promover a sobrevivência de uma célula do sistema nervoso, usos de um antagonista dr6 e métodos in vitro de inibir a ligação de dr6 com p75 |
-
2011
- 2011-06-07 EP EP11726021.6A patent/EP2580238A1/en not_active Withdrawn
- 2011-06-07 MX MX2012013899A patent/MX2012013899A/es active IP Right Grant
- 2011-06-07 CN CN201180039006.4A patent/CN103168048B/zh active Active
- 2011-06-07 CA CA2802017A patent/CA2802017A1/en not_active Abandoned
- 2011-06-07 WO PCT/US2011/039422 patent/WO2011156356A1/en active Application Filing
- 2011-06-07 BR BR112012031329A patent/BR112012031329A2/pt not_active Application Discontinuation
- 2011-06-07 JP JP2013514296A patent/JP2013531982A/ja active Pending
- 2011-06-07 US US13/701,234 patent/US8822642B2/en active Active
- 2011-06-07 EA EA201270802A patent/EA022932B1/ru not_active IP Right Cessation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006124667A2 (en) * | 2005-05-12 | 2006-11-23 | Zymogenetics, Inc. | Compositions and methods for modulating immune responses |
WO2009046407A2 (en) * | 2007-10-04 | 2009-04-09 | Zymogenetics, Inc. | B7 FAMILY MEMBER zB7H6 AND RELATED COMPOSITIONS AND METHODS |
Non-Patent Citations (1)
Title |
---|
THE SURFACE PROTEIN TIGIT SUPRESSES T CELL ACTIVATION BY PROMOTING THE GENERATION OF MATURE IMMUNOREGULATORY DENDRITIC CELLS;YU X ET AL;《NATURE IMMUNOLOGY》;20090101;第10卷(第1期);第48-57页 * |
Also Published As
Publication number | Publication date |
---|---|
US20130095102A1 (en) | 2013-04-18 |
EP2580238A1 (en) | 2013-04-17 |
CN103168048A (zh) | 2013-06-19 |
WO2011156356A1 (en) | 2011-12-15 |
CA2802017A1 (en) | 2011-12-15 |
MX2012013899A (es) | 2013-03-20 |
JP2013531982A (ja) | 2013-08-15 |
EA022932B1 (ru) | 2016-03-31 |
BR112012031329A2 (pt) | 2016-10-11 |
US8822642B2 (en) | 2014-09-02 |
EA201270802A1 (ru) | 2013-04-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103168048B (zh) | 二聚vstm3融合蛋白和相关的组合物和方法 | |
AU2021200367C1 (en) | Compositions and methods for adjoining type i and type ii extracellular domains as heterologous chimeric proteins | |
KR102392142B1 (ko) | 다량체 il-15 기반 분자 | |
KR102619015B1 (ko) | 면역 반응을 조절하는 방법 | |
JP2023075185A (ja) | 治療用ヌクレアーゼ組成物および方法 | |
CN110214147A (zh) | IL15/IL15Rα异源二聚体FC-融合蛋白 | |
KR102649757B1 (ko) | 5t4 및 4-1bb에 결합하는 항원 결합 단백질 및 관련 조성물 및 방법 | |
KR20110044991A (ko) | TNF-α 길항제 다-표적 결합 단백질 | |
CN101679497A (zh) | 可溶性il-17ra/rc融合蛋白以及相关方法 | |
HU228137B1 (en) | Soluble ctla4 mutant molecules and uses thereof | |
BRPI0616589A2 (pt) | polipeptìdeo, e, métodos para produzir um anticorpo para um polipeptìdeo, para reduzir ou inibir a inflamação e para tratar um mamìfero afligido com uma doença inflamatória | |
CN102770458A (zh) | 用于增加Fc融合蛋白的血清半寿期的组合物和方法 | |
JP2022514702A (ja) | 二機能性抗pd-1/il-7分子 | |
TW202136287A (zh) | 包含il-7變體之雙官能分子 | |
KR20230029622A (ko) | April 및 baff 억제 면역조절 단백질 및 사용 방법 | |
US20230303648A1 (en) | Bifunctional molecules comprising an il-7 variant | |
CN116848140A (zh) | 双功能抗pd1/il-7分子 | |
CN101316861A (zh) | Il-17a和il-17f拮抗剂以及使用所述拮抗剂的方法 | |
CN101514232B (zh) | 一种RANKL-Fc融合蛋白及其制备方法和用途 | |
US20240067727A1 (en) | Bifunctional anti-pd1/il-7 molecules | |
US20240182571A1 (en) | New scaffold for bifunctional molecules with improved properties | |
RU2804458C2 (ru) | Антигенсвязывающие белки, связывающиеся с 5t4 и 4-1bb, и относящиеся к ним композиции и способы | |
JP7278623B2 (ja) | 抗cd27抗体およびその使用 | |
KR20240053675A (ko) | sBCMA 변이체 및 이의 FC 융합 단백질을 이용한 IgA, IgM 및/또는 IgG의 생산 감소 방법 | |
CN116528889A (zh) | 用于治疗胶质母细胞瘤的方法和组合物 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant |