WO2017043569A1 - 新規なヒト血清アルブミン変異体 - Google Patents
新規なヒト血清アルブミン変異体 Download PDFInfo
- Publication number
- WO2017043569A1 WO2017043569A1 PCT/JP2016/076438 JP2016076438W WO2017043569A1 WO 2017043569 A1 WO2017043569 A1 WO 2017043569A1 JP 2016076438 W JP2016076438 W JP 2016076438W WO 2017043569 A1 WO2017043569 A1 WO 2017043569A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- protein
- serum albumin
- human serum
- hsa
- Prior art date
Links
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 274
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 274
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 245
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 171
- 235000018102 proteins Nutrition 0.000 claims abstract description 165
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 125
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 72
- 210000004369 blood Anatomy 0.000 claims abstract description 41
- 239000008280 blood Substances 0.000 claims abstract description 41
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 claims abstract description 7
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 6
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 99
- 239000013604 expression vector Substances 0.000 claims description 63
- 229920001184 polypeptide Polymers 0.000 claims description 63
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 63
- 208000020221 Short stature Diseases 0.000 claims description 41
- 108010051696 Growth Hormone Proteins 0.000 claims description 39
- 239000000122 growth hormone Substances 0.000 claims description 39
- 239000003814 drug Substances 0.000 claims description 35
- 210000004962 mammalian cell Anatomy 0.000 claims description 21
- 230000028327 secretion Effects 0.000 claims description 15
- 239000013598 vector Substances 0.000 claims description 15
- 230000001766 physiological effect Effects 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 229940088598 enzyme Drugs 0.000 claims description 13
- -1 pancreosimimine Chemical compound 0.000 claims description 13
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 claims description 12
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 claims description 12
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 12
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 12
- 229940112869 bone morphogenetic protein Drugs 0.000 claims description 12
- 206010056438 Growth hormone deficiency Diseases 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 230000007812 deficiency Effects 0.000 claims description 11
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 10
- 230000035772 mutation Effects 0.000 claims description 10
- LIFNDDBLJFPEAN-BPSSIEEOSA-N (2s)-4-amino-2-[[(2s)-2-[[2-[[2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-5-oxopyrrolidine-2-carbonyl]amino]propanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@@H]1CCC(=O)N1 LIFNDDBLJFPEAN-BPSSIEEOSA-N 0.000 claims description 9
- 101710185318 Thymic factor Proteins 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 9
- 108010025020 Nerve Growth Factor Proteins 0.000 claims description 8
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 claims description 8
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 claims description 8
- 239000012679 serum free medium Substances 0.000 claims description 8
- 229940124597 therapeutic agent Drugs 0.000 claims description 8
- 229960000187 tissue plasminogen activator Drugs 0.000 claims description 8
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 7
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims description 7
- 239000000275 Adrenocorticotropic Hormone Substances 0.000 claims description 6
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 claims description 6
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 claims description 6
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 claims description 6
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 claims description 6
- 102400000739 Corticotropin Human genes 0.000 claims description 6
- 101800000414 Corticotropin Proteins 0.000 claims description 6
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 6
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 6
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 claims description 6
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 6
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 6
- 102000004877 Insulin Human genes 0.000 claims description 6
- 102000009151 Luteinizing Hormone Human genes 0.000 claims description 6
- 108010073521 Luteinizing Hormone Proteins 0.000 claims description 6
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 6
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 claims description 6
- 102000015336 Nerve Growth Factor Human genes 0.000 claims description 6
- 108090000099 Neurotrophin-4 Proteins 0.000 claims description 6
- 102000003982 Parathyroid hormone Human genes 0.000 claims description 6
- 108090000445 Parathyroid hormone Proteins 0.000 claims description 6
- 102000004576 Placental Lactogen Human genes 0.000 claims description 6
- 108010003044 Placental Lactogen Proteins 0.000 claims description 6
- 239000000381 Placental Lactogen Substances 0.000 claims description 6
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims description 6
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims description 6
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 6
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 6
- 102000011923 Thyrotropin Human genes 0.000 claims description 6
- 108010061174 Thyrotropin Proteins 0.000 claims description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 6
- 208000026928 Turner syndrome Diseases 0.000 claims description 6
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 6
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 6
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 6
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 claims description 6
- 102400000015 Vasoactive intestinal peptide Human genes 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 6
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 claims description 6
- 210000000845 cartilage Anatomy 0.000 claims description 6
- 208000020832 chronic kidney disease Diseases 0.000 claims description 6
- 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 claims description 6
- 229960000258 corticotropin Drugs 0.000 claims description 6
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 6
- 229940040129 luteinizing hormone Drugs 0.000 claims description 6
- 229940053128 nerve growth factor Drugs 0.000 claims description 6
- 239000000199 parathyroid hormone Substances 0.000 claims description 6
- 229960001319 parathyroid hormone Drugs 0.000 claims description 6
- 102000003390 tumor necrosis factor Human genes 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- 208000030507 AIDS Diseases 0.000 claims description 5
- 108090001061 Insulin Proteins 0.000 claims description 5
- 229940125396 insulin Drugs 0.000 claims description 5
- 102000003951 Erythropoietin Human genes 0.000 claims description 4
- 102000006992 Interferon-alpha Human genes 0.000 claims description 4
- 108010047761 Interferon-alpha Proteins 0.000 claims description 4
- 201000010769 Prader-Willi syndrome Diseases 0.000 claims description 4
- 230000010261 cell growth Effects 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 229940105423 erythropoietin Drugs 0.000 claims description 4
- 230000002132 lysosomal effect Effects 0.000 claims description 4
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 4
- LVRVABPNVHYXRT-BQWXUCBYSA-N 52906-92-0 Chemical compound C([C@H](N)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)C1=CC=CC=C1 LVRVABPNVHYXRT-BQWXUCBYSA-N 0.000 claims description 3
- 102000006772 Acid Ceramidase Human genes 0.000 claims description 3
- 108020005296 Acid Ceramidase Proteins 0.000 claims description 3
- 108010059616 Activins Proteins 0.000 claims description 3
- 102000005606 Activins Human genes 0.000 claims description 3
- 102100034561 Alpha-N-acetylglucosaminidase Human genes 0.000 claims description 3
- 102100040894 Amylo-alpha-1,6-glucosidase Human genes 0.000 claims description 3
- 102400000068 Angiostatin Human genes 0.000 claims description 3
- 108010079709 Angiostatins Proteins 0.000 claims description 3
- 108010064733 Angiotensins Proteins 0.000 claims description 3
- 102000015427 Angiotensins Human genes 0.000 claims description 3
- 108010024976 Asparaginase Proteins 0.000 claims description 3
- 102000015790 Asparaginase Human genes 0.000 claims description 3
- 108010023546 Aspartylglucosylaminase Proteins 0.000 claims description 3
- 108010001478 Bacitracin Proteins 0.000 claims description 3
- 101710124976 Beta-hexosaminidase A Proteins 0.000 claims description 3
- 101710124978 Beta-hexosaminidase B Proteins 0.000 claims description 3
- 102100032487 Beta-mannosidase Human genes 0.000 claims description 3
- 108010051479 Bombesin Proteins 0.000 claims description 3
- 102000013585 Bombesin Human genes 0.000 claims description 3
- 101800004538 Bradykinin Proteins 0.000 claims description 3
- 102400000967 Bradykinin Human genes 0.000 claims description 3
- 108060001064 Calcitonin Proteins 0.000 claims description 3
- 102000055006 Calcitonin Human genes 0.000 claims description 3
- 108010010737 Ceruletide Proteins 0.000 claims description 3
- 101800001982 Cholecystokinin Proteins 0.000 claims description 3
- 102100025841 Cholecystokinin Human genes 0.000 claims description 3
- 241000511343 Chondrostoma nasus Species 0.000 claims description 3
- 108010078777 Colistin Proteins 0.000 claims description 3
- 108010065372 Dynorphins Proteins 0.000 claims description 3
- 102000009025 Endorphins Human genes 0.000 claims description 3
- 108010049140 Endorphins Proteins 0.000 claims description 3
- 102400001047 Endostatin Human genes 0.000 claims description 3
- 108010079505 Endostatins Proteins 0.000 claims description 3
- 108010092674 Enkephalins Proteins 0.000 claims description 3
- 108090000394 Erythropoietin Proteins 0.000 claims description 3
- 108010076282 Factor IX Proteins 0.000 claims description 3
- 108010023321 Factor VII Proteins 0.000 claims description 3
- 108010054218 Factor VIII Proteins 0.000 claims description 3
- 102000001690 Factor VIII Human genes 0.000 claims description 3
- 102100028496 Galactocerebrosidase Human genes 0.000 claims description 3
- 108010042681 Galactosylceramidase Proteins 0.000 claims description 3
- 102400000921 Gastrin Human genes 0.000 claims description 3
- 108010052343 Gastrins Proteins 0.000 claims description 3
- 102400000321 Glucagon Human genes 0.000 claims description 3
- 108060003199 Glucagon Proteins 0.000 claims description 3
- 102000004547 Glucosylceramidase Human genes 0.000 claims description 3
- 108010017544 Glucosylceramidase Proteins 0.000 claims description 3
- 102000053187 Glucuronidase Human genes 0.000 claims description 3
- 108010060309 Glucuronidase Proteins 0.000 claims description 3
- YWAQATDNEKZFFK-BYPYZUCNSA-N Gly-Gly-Ser Chemical compound NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O YWAQATDNEKZFFK-BYPYZUCNSA-N 0.000 claims description 3
- 108010026389 Gramicidin Proteins 0.000 claims description 3
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 claims description 3
- 229920002971 Heparan sulfate Polymers 0.000 claims description 3
- 102000004627 Iduronidase Human genes 0.000 claims description 3
- 108010003381 Iduronidase Proteins 0.000 claims description 3
- 102000003996 Interferon-beta Human genes 0.000 claims description 3
- 108090000467 Interferon-beta Proteins 0.000 claims description 3
- 102000008070 Interferon-gamma Human genes 0.000 claims description 3
- 108010074328 Interferon-gamma Proteins 0.000 claims description 3
- 102100020873 Interleukin-2 Human genes 0.000 claims description 3
- 108010002350 Interleukin-2 Proteins 0.000 claims description 3
- 108060005987 Kallikrein Proteins 0.000 claims description 3
- 102000001399 Kallikrein Human genes 0.000 claims description 3
- 101710163560 Lamina-associated polypeptide 2, isoform alpha Proteins 0.000 claims description 3
- 101710189385 Lamina-associated polypeptide 2, isoforms beta/gamma Proteins 0.000 claims description 3
- 102000016267 Leptin Human genes 0.000 claims description 3
- 108010092277 Leptin Proteins 0.000 claims description 3
- URLZCHNOLZSCCA-VABKMULXSA-N Leu-enkephalin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 URLZCHNOLZSCCA-VABKMULXSA-N 0.000 claims description 3
- 239000000637 Melanocyte-Stimulating Hormone Substances 0.000 claims description 3
- 108010007013 Melanocyte-Stimulating Hormones Proteins 0.000 claims description 3
- 101800002372 Motilin Proteins 0.000 claims description 3
- 102100021003 N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase Human genes 0.000 claims description 3
- 101800001814 Neurotensin Proteins 0.000 claims description 3
- 102400001103 Neurotensin Human genes 0.000 claims description 3
- 102000004230 Neurotrophin 3 Human genes 0.000 claims description 3
- 108090000742 Neurotrophin 3 Proteins 0.000 claims description 3
- 102000003683 Neurotrophin-4 Human genes 0.000 claims description 3
- 102100033857 Neurotrophin-4 Human genes 0.000 claims description 3
- 108090000095 Neurotrophin-6 Proteins 0.000 claims description 3
- 102400000050 Oxytocin Human genes 0.000 claims description 3
- 101800000989 Oxytocin Proteins 0.000 claims description 3
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 claims description 3
- 108010093965 Polymyxin B Proteins 0.000 claims description 3
- 102100024622 Proenkephalin-B Human genes 0.000 claims description 3
- 108010057464 Prolactin Proteins 0.000 claims description 3
- 108050007079 Saposin Proteins 0.000 claims description 3
- 102000017852 Saposin Human genes 0.000 claims description 3
- 108010086019 Secretin Proteins 0.000 claims description 3
- 102100037505 Secretin Human genes 0.000 claims description 3
- 108010056088 Somatostatin Proteins 0.000 claims description 3
- 102000005157 Somatostatin Human genes 0.000 claims description 3
- 102000036693 Thrombopoietin Human genes 0.000 claims description 3
- 108010041111 Thrombopoietin Proteins 0.000 claims description 3
- 102400000159 Thymopoietin Human genes 0.000 claims description 3
- 239000000898 Thymopoietin Substances 0.000 claims description 3
- 108010046075 Thymosin Proteins 0.000 claims description 3
- 102000007501 Thymosin Human genes 0.000 claims description 3
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 claims description 3
- 108010044965 UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosaminephosphotransferase Proteins 0.000 claims description 3
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 claims description 3
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims description 3
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 claims description 3
- 108010004977 Vasopressins Proteins 0.000 claims description 3
- 102000002852 Vasopressins Human genes 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000000488 activin Substances 0.000 claims description 3
- 108010030291 alpha-Galactosidase Proteins 0.000 claims description 3
- 102000005840 alpha-Galactosidase Human genes 0.000 claims description 3
- 102000012086 alpha-L-Fucosidase Human genes 0.000 claims description 3
- 108010061314 alpha-L-Fucosidase Proteins 0.000 claims description 3
- 108010012864 alpha-Mannosidase Proteins 0.000 claims description 3
- 102000019199 alpha-Mannosidase Human genes 0.000 claims description 3
- 102000002014 alpha-N-Acetylgalactosaminidase Human genes 0.000 claims description 3
- 108010015684 alpha-N-Acetylgalactosaminidase Proteins 0.000 claims description 3
- 108010009380 alpha-N-acetyl-D-glucosaminidase Proteins 0.000 claims description 3
- 108010006759 amylo-1,6-glucosidase Proteins 0.000 claims description 3
- 208000022531 anorexia Diseases 0.000 claims description 3
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 claims description 3
- 229960003272 asparaginase Drugs 0.000 claims description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 claims description 3
- 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 claims description 3
- 229960003071 bacitracin Drugs 0.000 claims description 3
- 229930184125 bacitracin Natural products 0.000 claims description 3
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 3
- 108010055059 beta-Mannosidase Proteins 0.000 claims description 3
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 claims description 3
- 229960004015 calcitonin Drugs 0.000 claims description 3
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 3
- YRALAIOMGQZKOW-HYAOXDFASA-N ceruletide Chemical compound C([C@@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)[C@@H](C)O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(OS(O)(=O)=O)C=C1 YRALAIOMGQZKOW-HYAOXDFASA-N 0.000 claims description 3
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 claims description 3
- 229940107137 cholecystokinin Drugs 0.000 claims description 3
- 229960003346 colistin Drugs 0.000 claims description 3
- 206010061428 decreased appetite Diseases 0.000 claims description 3
- 230000004069 differentiation Effects 0.000 claims description 3
- 230000027119 gastric acid secretion Effects 0.000 claims description 3
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 3
- 229960004666 glucagon Drugs 0.000 claims description 3
- 108010054451 glucosamine acetyltransferase Proteins 0.000 claims description 3
- 229960004905 gramicidin Drugs 0.000 claims description 3
- ZWCXYZRRTRDGQE-SORVKSEFSA-N gramicidina Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CC=3C4=CC=CC=C4NC=3)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@H](NC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](NC=O)C(C)C)CC(C)C)C(=O)NCCO)=CNC2=C1 ZWCXYZRRTRDGQE-SORVKSEFSA-N 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 229960003130 interferon gamma Drugs 0.000 claims description 3
- 229960001388 interferon-beta Drugs 0.000 claims description 3
- 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 claims description 3
- 229940039781 leptin Drugs 0.000 claims description 3
- 239000003446 ligand Substances 0.000 claims description 3
- 210000003593 megakaryocyte Anatomy 0.000 claims description 3
- CWWARWOPSKGELM-SARDKLJWSA-N methyl (2s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-5-amino-2-[[(2s)-1-[(2s)-6-amino-2-[[(2s)-1-[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-5 Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)OC)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 CWWARWOPSKGELM-SARDKLJWSA-N 0.000 claims description 3
- JORAUNFTUVJTNG-BSTBCYLQSA-N n-[(2s)-4-amino-1-[[(2s,3r)-1-[[(2s)-4-amino-1-oxo-1-[[(3s,6s,9s,12s,15r,18s,21s)-6,9,18-tris(2-aminoethyl)-3-[(1r)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-h Chemical compound CC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O.CCC(C)CCCCC(=O)N[C@@H](CCN)C(=O)N[C@H]([C@@H](C)O)CN[C@@H](CCN)C(=O)N[C@H]1CCNC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O JORAUNFTUVJTNG-BSTBCYLQSA-N 0.000 claims description 3
- PCJGZPGTCUMMOT-ISULXFBGSA-N neurotensin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 PCJGZPGTCUMMOT-ISULXFBGSA-N 0.000 claims description 3
- 229940032018 neurotrophin 3 Drugs 0.000 claims description 3
- 229940097998 neurotrophin 4 Drugs 0.000 claims description 3
- 229960001723 oxytocin Drugs 0.000 claims description 3
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 claims description 3
- 229920000024 polymyxin B Polymers 0.000 claims description 3
- XDJYMJULXQKGMM-UHFFFAOYSA-N polymyxin E1 Natural products CCC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O XDJYMJULXQKGMM-UHFFFAOYSA-N 0.000 claims description 3
- KNIWPHSUTGNZST-UHFFFAOYSA-N polymyxin E2 Natural products CC(C)CCCCC(=O)NC(CCN)C(=O)NC(C(C)O)C(=O)NC(CCN)C(=O)NC1CCNC(=O)C(C(C)O)NC(=O)C(CCN)NC(=O)C(CCN)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CCN)NC1=O KNIWPHSUTGNZST-UHFFFAOYSA-N 0.000 claims description 3
- 229960005266 polymyxin b Drugs 0.000 claims description 3
- 229940097325 prolactin Drugs 0.000 claims description 3
- 239000003488 releasing hormone Substances 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 229960002101 secretin Drugs 0.000 claims description 3
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)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](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)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)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 claims description 3
- 230000003248 secreting effect Effects 0.000 claims description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 claims description 3
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 claims description 3
- 229960000553 somatostatin Drugs 0.000 claims description 3
- 210000000130 stem cell Anatomy 0.000 claims description 3
- YRALAIOMGQZKOW-UHFFFAOYSA-N sulfated caerulein Natural products C=1C=CC=CC=1CC(C(N)=O)NC(=O)C(CC(O)=O)NC(=O)C(CCSC)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(C(C)O)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CCC(N)=O)NC(=O)C1NC(=O)CC1)CC1=CC=C(OS(O)(=O)=O)C=C1 YRALAIOMGQZKOW-UHFFFAOYSA-N 0.000 claims description 3
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([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](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)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](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 claims description 3
- 239000005495 thyroid hormone Substances 0.000 claims description 3
- 229940036555 thyroid hormone Drugs 0.000 claims description 3
- 229960005356 urokinase Drugs 0.000 claims description 3
- 229960003726 vasopressin Drugs 0.000 claims description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 2
- 102000007072 Nerve Growth Factors Human genes 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 239000003900 neurotrophic factor Substances 0.000 claims description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 2
- 102100038803 Somatotropin Human genes 0.000 claims 6
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims 2
- 102000002419 Motilin Human genes 0.000 claims 1
- 102000016943 Muramidase Human genes 0.000 claims 1
- 108010014251 Muramidase Proteins 0.000 claims 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims 1
- 108010023320 N-acetylglucosamine-6-sulfatase Proteins 0.000 claims 1
- 102000003946 Prolactin Human genes 0.000 claims 1
- 102000013275 Somatomedins Human genes 0.000 claims 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 claims 1
- 210000002919 epithelial cell Anatomy 0.000 claims 1
- 229960000274 lysozyme Drugs 0.000 claims 1
- 235000010335 lysozyme Nutrition 0.000 claims 1
- 239000004325 lysozyme Substances 0.000 claims 1
- 210000004498 neuroglial cell Anatomy 0.000 claims 1
- 230000002992 thymic effect Effects 0.000 claims 1
- 230000000975 bioactive effect Effects 0.000 abstract description 4
- 108010000521 Human Growth Hormone Proteins 0.000 description 103
- 239000000854 Human Growth Hormone Substances 0.000 description 102
- 102000002265 Human Growth Hormone Human genes 0.000 description 101
- 210000004027 cell Anatomy 0.000 description 99
- 102000037865 fusion proteins Human genes 0.000 description 57
- 108020001507 fusion proteins Proteins 0.000 description 57
- 108020004414 DNA Proteins 0.000 description 48
- 239000012634 fragment Substances 0.000 description 36
- 102000018997 Growth Hormone Human genes 0.000 description 33
- 230000014509 gene expression Effects 0.000 description 30
- 229920001223 polyethylene glycol Polymers 0.000 description 30
- 239000002202 Polyethylene glycol Substances 0.000 description 28
- 239000002609 medium Substances 0.000 description 28
- 101001075287 Homo sapiens Growth hormone receptor Proteins 0.000 description 27
- 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 26
- 241000699666 Mus <mouse, genus> Species 0.000 description 25
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 24
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 23
- 235000001014 amino acid Nutrition 0.000 description 23
- 229940024606 amino acid Drugs 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 21
- 238000002360 preparation method Methods 0.000 description 21
- 229940079593 drug Drugs 0.000 description 19
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 18
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 18
- 210000004899 c-terminal region Anatomy 0.000 description 18
- 230000001965 increasing effect Effects 0.000 description 18
- 239000003550 marker Substances 0.000 description 18
- 238000010276 construction Methods 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 108091008146 restriction endonucleases Proteins 0.000 description 17
- 241000282567 Macaca fascicularis Species 0.000 description 16
- 230000000694 effects Effects 0.000 description 16
- 241000710188 Encephalomyocarditis virus Species 0.000 description 14
- 241000282414 Homo sapiens Species 0.000 description 14
- 241001465754 Metazoa Species 0.000 description 13
- 102000005396 glutamine synthetase Human genes 0.000 description 13
- 108020002326 glutamine synthetase Proteins 0.000 description 13
- 229950010131 puromycin Drugs 0.000 description 13
- 108010022394 Threonine synthase Proteins 0.000 description 12
- SXTAYKAGBXMACB-UHFFFAOYSA-N methionine S-imide-S-oxide Natural products CS(=N)(=O)CCC(N)C(O)=O SXTAYKAGBXMACB-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 206010059866 Drug resistance Diseases 0.000 description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 10
- 102000004419 dihydrofolate reductase Human genes 0.000 description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 9
- 239000004473 Threonine Substances 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000006467 substitution reaction Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000037396 body weight Effects 0.000 description 8
- 230000004663 cell proliferation Effects 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 7
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 7
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 7
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 206010041092 Small for dates baby Diseases 0.000 description 6
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 6
- 125000000524 functional group Chemical group 0.000 description 6
- 230000008488 polyadenylation Effects 0.000 description 6
- 239000006152 selective media Substances 0.000 description 6
- 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 5
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- 108091081024 Start codon Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 235000004279 alanine Nutrition 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 230000037356 lipid metabolism Effects 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000010254 subcutaneous injection Methods 0.000 description 5
- 239000007929 subcutaneous injection Substances 0.000 description 5
- 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 4
- XEGZSHSPQNDNRH-JRQIVUDYSA-N Asn-Tyr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XEGZSHSPQNDNRH-JRQIVUDYSA-N 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 4
- 229930193140 Neomycin Natural products 0.000 description 4
- 108700005078 Synthetic Genes Proteins 0.000 description 4
- 108010054737 albumin Redhill Proteins 0.000 description 4
- 239000003638 chemical reducing agent Substances 0.000 description 4
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 4
- 229960004927 neomycin Drugs 0.000 description 4
- 229920001451 polypropylene glycol Polymers 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 3
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 239000004372 Polyvinyl alcohol Substances 0.000 description 3
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 229960002449 glycine Drugs 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 description 3
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 3
- 239000013587 production medium Substances 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000008215 water for injection Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- UVGHPGOONBRLCX-NJSLBKSFSA-N (2,5-dioxopyrrolidin-1-yl) 6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)NCCCCCC(=O)ON1C(=O)CCC1=O UVGHPGOONBRLCX-NJSLBKSFSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- IEJPPSMHUUQABK-UHFFFAOYSA-N 2,4-diphenyl-4h-1,3-oxazol-5-one Chemical compound O=C1OC(C=2C=CC=CC=2)=NC1C1=CC=CC=C1 IEJPPSMHUUQABK-UHFFFAOYSA-N 0.000 description 2
- QXZBMSIDSOZZHK-DOPDSADYSA-N 31362-50-2 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CNC=N1 QXZBMSIDSOZZHK-DOPDSADYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 229920002101 Chitin Polymers 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 108020005350 Initiator Codon Proteins 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- 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 2
- 241000124008 Mammalia Species 0.000 description 2
- 102400001357 Motilin Human genes 0.000 description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 102100024819 Prolactin Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 102000005262 Sulfatase Human genes 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000019445 benzyl alcohol Nutrition 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 230000022159 cartilage development Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- PYLIXCKOHOHGKQ-UHFFFAOYSA-L disodium;hydrogen phosphate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O PYLIXCKOHOHGKQ-UHFFFAOYSA-L 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- CEIPQQODRKXDSB-UHFFFAOYSA-N ethyl 3-(6-hydroxynaphthalen-2-yl)-1H-indazole-5-carboximidate dihydrochloride Chemical compound Cl.Cl.C1=C(O)C=CC2=CC(C3=NNC4=CC=C(C=C43)C(=N)OCC)=CC=C21 CEIPQQODRKXDSB-UHFFFAOYSA-N 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 229960000890 hydrocortisone Drugs 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- LFKYBJLFJOOKAE-UHFFFAOYSA-N imidazol-2-ylidenemethanone Chemical compound O=C=C1N=CC=N1 LFKYBJLFJOOKAE-UHFFFAOYSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000012948 isocyanate Substances 0.000 description 2
- 150000002513 isocyanates Chemical class 0.000 description 2
- 150000002540 isothiocyanates Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940073475 lysozyme hydrochloride Drugs 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229920001583 poly(oxyethylated polyols) Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 229920001289 polyvinyl ether Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 235000004400 serine Nutrition 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 108060007951 sulfatase Proteins 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 2
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101150029662 E1 gene Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 101150074355 GS gene Proteins 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 241000709721 Hepatovirus A Species 0.000 description 1
- 101000760602 Homo sapiens ATP-binding cassette sub-family D member 1 Proteins 0.000 description 1
- 101000899240 Homo sapiens Endoplasmic reticulum chaperone BiP Proteins 0.000 description 1
- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 description 1
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 description 1
- 101000788682 Homo sapiens GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 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
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091006006 PEGylated Proteins Proteins 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102100040918 Pro-glucagon Human genes 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000012327 Ruthenium complex Substances 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 241001575928 Siler Species 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001294 alanine derivatives Chemical class 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 210000004198 anterior pituitary gland Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 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
- 230000033228 biological regulation Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- GHWVXCQZPNWFRO-UHFFFAOYSA-N butane-2,3-diamine Chemical compound CC(N)C(C)N GHWVXCQZPNWFRO-UHFFFAOYSA-N 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 102000047168 human ABCD1 Human genes 0.000 description 1
- 102000044162 human IGF1 Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 150000002506 iron compounds Chemical class 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000007180 physiological regulation Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- UWCDGNNRRPICBV-FROKLYQUSA-N sulfuric acid N-[(3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound OS(O)(=O)=O.CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O UWCDGNNRRPICBV-FROKLYQUSA-N 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229960003873 thymostimulin Drugs 0.000 description 1
- 230000002916 thymostimulin Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH], i.e. somatotropin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Definitions
- the present invention relates to a novel human serum albumin mutant capable of enhancing the blood stability of the protein by linking with a protein having physiological activity, and the human serum albumin mutant and a protein having physiological activity.
- the present invention relates to a linked human serum albumin mutant-linked protein (HSA mutant-linked protein), for example, human serum albumin mutant-linked human growth hormone.
- HSA Human serum albumin
- HSA Human serum albumin
- human serum albumin has multiple natural variants.
- Human serum albumin Redhill is one of them (Non-Patent Documents 1 and 2).
- Non-Patent Documents 1 and 2 Compared to the amino acid sequence of the above-mentioned normal human serum albumin consisting of 585 amino acids, human serum albumin Redhill has threonine instead of alanine at the 320th amino acid residue from the N-terminal side. It differs in that one arginine residue is added, and it consists of 586 amino acids.
- albumin Redhill Due to the above change of alanine to threonine, albumin Redhill generates a sequence represented by Asn-Tyr-Thr in its amino acid sequence, and the Asn (asparagine) residue in this sequence is N-linked glycosidated. For this reason, albumin Redhill is observed to have a molecular weight of about 2.5 kDa larger than that of the normal human serum albumin.
- Non-patent Documents 3, Patent Documents 1 and 2 A method for increasing the stability of a protein such as an enzyme in plasma by fusing HSA to the protein has been reported (Non-patent Documents 3, Patent Documents 1 and 2).
- a fusion protein of HSA and an enzyme or the like is prepared by preparing a transformed cell into which an expression vector incorporating a DNA in which a gene encoding a protein such as HSA and a gene encoding a protein such as an enzyme are linked in frame is introduced. By culturing the cells, it is produced as a recombinant protein in the medium or in the cells.
- proteins fused with human serum albumin (HSA) to increase plasma stability include fusion proteins of HSA and G-CSF (Patent Documents 1 and 3), fusion of HSA and interferon ⁇ Protein (patent document 4), fusion protein of HSA and GLP-1 (patent document 5), fusion protein of HSA and insulin (patent document 6), fusion protein of HSA and erythropoietin (patent document 7), HSA and Examples include fusion proteins with growth hormone (Patent Documents 4, 5, and 8 to 11).
- hGH Human growth hormone
- a preparation (hGH preparation) containing hGH with a molecular weight of about 22 KD as an active ingredient, produced as a recombinant protein using Escherichia coli introduced with the hGH gene, is a growth hormone secretory short stature, short stature in Turner syndrome, bone Widely used as a therapeutic agent for SGA (Small-for-Gestational Age) short stature without end-line closure, short stature due to chronic renal failure, short stature in Praderwillie syndrome, short stature in cartilage dystrophy Applied.
- the hGH preparation is administered subcutaneously or intramuscularly, circulates in the blood, and has an effect of promoting the growth of the patient by its growth promoting activity.
- hGH preparations are widely applied clinically as therapeutic agents for adult growth hormone secretion deficiency.
- various abnormalities such as abnormal lipid metabolism are observed, but administration of the hGH preparation improves the patient's QOL, such as normalizing the patient's lipid metabolism.
- Examples of hGH preparations for growth hormone secretion deficiency short stature, adult growth hormone secretion deficiency and the like include GLOJECT (registered trademark).
- the attempt to increase the stability of hGH in plasma is due to clinical requirements.
- the half-life of hGH in plasma is less than 20 minutes, and hGH administered to a patient disappears rapidly from the blood. For this reason, in order for patients to substantially exert the efficacy of hGH, it is necessary to administer hGH three times a week intramuscularly or subcutaneously every day. Such frequent administration is a burden on the patient. Therefore, it is preferable that the burden on the patient can be reduced if the number of administrations of hGH to the patient can be reduced by increasing the stability of hGH in plasma and extending the half-life.
- JP 7-503368 Japanese Patent Laid-Open No. 3-178998 JP 7-503844
- an object of the present invention is to increase the blood stability of a physiologically active protein by binding it to a desired physiologically active protein (also referred to as protein (A) in the present specification).
- a desired physiologically active protein also referred to as protein (A) in the present specification.
- a further object of the present invention is to provide a human serum albumin variant-linked protein comprising a desired protein (eg, growth hormone) and the human serum albumin variant linked thereto.
- a still further object of the present invention is to provide a method for increasing the blood stability of the protein by linking the desired protein with the human serum albumin variant.
- arginine which is the 320th amino acid residue from the N-terminal, is threonine compared to normal human serum albumin consisting of 585 amino acids.
- a compound (human serum albumin mutant-linked hGH) obtained by linking a mutant (human serum albumin mutant) consisting of the amino acid sequence to be replaced with human growth hormone (hGH) was administered to a living body. Occasionally, it was found that the blood stability was significantly higher than that of the original human growth hormone, and further studies were continued to complete the present invention. That is, the present invention provides the following. 1.
- amino acid sequence is shown in SEQ ID NO: 3.
- a human serum albumin variant comprising an amino acid sequence that is conserved. 2. The human serum albumin variant according to 1 above, wherein the amino acid residue (X) is tyrosine. 3.
- the human serum albumin variant according to claim 2 comprising the amino acid sequence represented by SEQ ID NO: 3. 4).
- the amino acid sequence represented by SEQ ID NO: 3 is 10 or less except in the sequence portion corresponding to the position 318 to 320 from the N-terminus.
- a second polypeptide comprising the first polypeptide chain comprising the amino acid sequence of any one of the above-mentioned human serum albumin variants and the amino acid sequence of another protein (A) linked thereto; A human serum albumin mutant-linked protein (A), comprising a peptide chain. 7).
- the protein (A) is ⁇ -L-iduronidase, iduronic acid-2-sulfatase, glucocerebrosidase, ⁇ -galactosidase, GM2-activated protein, ⁇ -hexosaminidase A, ⁇ -hexosaminidase B, N -Acetylglucosamine-1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, galactosylceramidase, saposin C, allylsulfatase A, ⁇ -L-fucosidase, aspartylglucosaminidase, ⁇ -N-acetylgalactosaminidase, acid sphingomyeloid Nase, ⁇ -galactosidase, ⁇ -glucuronidase, heparan sulfate N-sulfatase, ⁇ -N-acetylgluco
- the drug according to 19 above which is a therapeutic agent for a disease selected from the group consisting of those without epiphyseal closure, and adult growth hormone secretion deficiency, exhaustion due to AIDS, and exhaustion due to anorexia.
- 21. DNA comprising a gene encoding the human serum albumin variant according to any one of 1 to 5 above.
- 22. DNA comprising a gene encoding the human serum albumin variant linking protein (A) of any one of 6 to 18 above.
- An expression vector comprising the DNA of 21 or 22 above.
- 24. A mammalian cell transformed with the above 23 vector.
- Human serum albumin mutant or human serum albumin mutant-linked protein (A) obtained by culturing the above 24 mammalian cells in a serum-free medium.
- the blood stability of a desired physiologically active protein having physiological activity to be administered to animals (including humans) as a pharmaceutical can be enhanced. Therefore, the medicinal efficacy of those bioactive proteins can be increased, and the duration of the medicinal efficacy can be extended. This also makes it possible to reduce the dose and frequency of administration of these physiologically active proteins, thereby improving the patient's QOL and contributing to the prevention of infection and medical accidents caused by conventional frequent administration. can do.
- the flowchart which shows the construction method of a pE-neo vector The flowchart which shows the construction method of a pE-hygr vector.
- the flowchart which shows the construction method of pE-IRES-GS-puro The flowchart which shows the construction method of pE-IRES-GS-puro.
- the flowchart which shows the construction method of pE-IRES-GS-puro The flowchart which shows the construction method of pE-IRES-GS-puro.
- the flowchart which shows the construction method of pE-IRES-GS-puro The flowchart which shows the construction method of pE-IRES-GS-puro.
- the flowchart which shows the construction method of pE-IRES-GS-puro The flowchart which shows the construction method of pE-IRES-GS-puro.
- the flowchart which shows the construction method of pE-IRES-GS-puro The flowchart which shows the construction method of pE-IRES-GS-puro.
- the vertical axis represents the absorbance at 490 nm, and the horizontal axis represents the concentration (nM) of each specimen. Vertical bars indicate standard deviation.
- the graph which shows the result of the pharmacokinetic analysis of HSA-hGH fusion protein using a cynomolgus monkey.
- the vertical axis represents the concentration (ng / mL) of HSA-hGH fusion protein in cynomolgus monkey plasma, and the horizontal axis represents the elapsed time (hours) after administration of the HSA-hGH fusion protein. Vertical bars in the graph indicate standard deviation.
- the graph which shows the result of a medicinal effect analysis of the HSA-hGH fusion protein using a cynomolgus monkey.
- the vertical axis is the concentration (%) of IGF-1 in cynomolgus monkey plasma when the concentration before administration of the HSA-hGH fusion protein is 100%, and the horizontal axis is the elapsed time (days) after administration of the HSA-hGH fusion protein. ). Vertical bars in the graph indicate standard deviation.
- human serum albumin refers to endogenous wild-type human serum albumin consisting of 585 amino acid residues represented by SEQ ID NO: 1 in addition to endogenous wild-type human serum albumin.
- SEQ ID NO: 1 As long as it has a function as a normal wild-type human serum albumin such as a function of binding and transporting exogenous substances such as substances and drugs, one or more of the amino acid sequence shown in SEQ ID NO: 1 Amino acid residues have been substituted, deleted, and / or added (in this specification, “addition” of amino acid residues means adding residues at the end or within the sequence). Mutants of HSA corresponding to those are also included without distinction.
- the number of amino acid residues to be substituted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3. is there.
- the number of amino acid residues to be deleted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3.
- a variant consisting of 584 amino acid residues from which the N-terminal or C-terminal amino acid residue of the amino acid sequence represented by SEQ ID NO: 1 has been deleted is also included in human serum albumin.
- amino acid residues are added to the amino acid sequence of normal wild-type HSA or a variant thereof, or to the N-terminal side or C-terminal side of the amino acid sequence (“addition” is the end of the sequence). Or it means adding a residue inside).
- the number of amino acid residues added at this time is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3.
- HSA mutants introduced by combining at least two kinds of mutations are 0-10 with respect to the amino acid sequence shown in SEQ ID NO: 1. Having an amino acid sequence obtained by deletion of amino acid residues, substitution of 0 to 10 amino acid residues with other amino acid residues, and addition of 0 to 10 amino acid residues preferable. More preferably, the number of amino acid residues to be deleted, substituted and / or added to the amino acid sequence represented by SEQ ID NO: 1 is preferably 5 or less, more preferably 3 or less, respectively.
- human serum albumin Redhill means a variant of human serum albumin consisting of 586 amino acid residues represented by SEQ ID NO: 2.
- Human serum albumin Redhill is threonine, not alanine, in the 320th amino acid residue from the N-terminal of the amino acid sequence of wild-type human serum albumin consisting of 585 amino acids shown in SEQ ID NO: 1 and N-terminal. Is equivalent to the addition of one arginine residue.
- albumin Redhill is observed to have a molecular weight of about 2.5 kDa larger than that of normal wild-type albumin (SEQ ID NO: 1).
- human serum albumin variant is the above-mentioned variant of normal wild-type HSA (SEQ ID NO: 1), provided that the variant (HSA ⁇ Other than Redhill).
- a preferred HSA variant in the present invention is a normal wild-type human serum albumin having a function of binding and transporting an endogenous substance in blood and an exogenous substance such as a drug in addition to that shown in SEQ ID NO: 3.
- amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 are substituted, deleted, or added to other amino acid residues
- the 318th asparagine residue and the 320th threonine residue from the N-terminal of the amino acid sequence shown in SEQ ID NO: 3 are a single amino acid residue other than proline between these two residues ( Including those having an amino acid sequence that is conserved in a linked state via a peptide bond via X).
- the number of amino acid residues to be substituted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3 pieces.
- the number of amino acid residues to be deleted is preferably 1 to 10, more preferably 1 to 5, and still more preferably 1 to 3.
- a variant consisting of 584 amino acid residues from which the N-terminal or C-terminal amino acid residue of the amino acid sequence represented by SEQ ID NO: 3 has been deleted may be used. Further, a combination of substitution and deletion of these amino acid residues may be used. Furthermore, one or a plurality of amino acid residues may be added to the amino acid sequences of these variants or to the N-terminal side or C-terminal side of the amino acid sequence.
- amino acid sequence represented by SEQ ID NO: 3 was introduced by combining at least two kinds of mutations among three kinds of mutations of amino acid substitution, deletion, and addition, and 0-10 Deletion of amino acid residues, substitution of 0 to 10 amino acid residues with other amino acid residues, and addition of 0 to 10 amino acid residues can be performed.
- amino acid residue at positions 318 to 320 from the N-terminal of the amino acid sequence shown in SEQ ID NO: 3 must be asparagine-X-threonine (“X” is an amino acid residue other than proline), preferably asparagine. -Tyrosine-threonine.
- the human serum albumin variant (a typical example of the HSA variant of the present invention) prepared in the examples described below is compared to the amino acid sequence (SEQ ID NO: 1) of wild-type human serum albumin consisting of 585 amino acids. It differs only in that the 320th amino acid residue from the N-terminal is not alanine but threonine (SEQ ID NO: 3). Due to this difference, the HSA mutant (referred to as “HSA320T)”) has a sequence part represented by Asn-Tyr-Thr in its amino acid sequence, and the Asn (asparagine) residue is N in this sequence part. -It can be linked glycosidated.
- the HSA mutant of the present invention is produced as a recombinant protein by preparing an expression vector incorporating the DNA encoding the HSA mutant of the present invention and culturing host cells transformed with this expression vector. can do.
- a protein having physiological activity to be linked to a human serum albumin variant is serum albumin (whether or not it is a variant).
- proteins other than having physiological activity.
- physiological activity refers to the ability to act on a living body to cause some specific physiological change. For example, various enzymes (for example, lysosomal enzymes), peptide hormones (protein hormones) , Neurotransmitters, growth factors, signaling factors, and other proteins involved in various physiological regulation (promotion and suppression).
- human serum albumin mutant-linked protein (A) or “HSA mutant-linked protein (A)” refers to the protein (A) to which the HSA mutant of the present invention is linked, This is a compound obtained by linking polypeptides having both amino acid sequences.
- “ligating” these polypeptides is not only based on a direct peptide bond between one N-terminus and the other C-terminus but also indirectly via a linker. Including the case of combining.
- the “linker” is a structural part that is between the two polypeptides and covalently links the two polypeptides, and is derived from any end of the HSA mutant of the present invention and the protein (A) that is the linking partner. It is something that does not.
- the linker can be a single amino acid residue or a peptide chain moiety consisting of two or more amino acid residues (peptide linker), which is peptide-bonded to both polypeptides, with one or more amino acid residues remaining.
- a linker consisting of a group is generically referred to herein as a “peptide linker”.
- the linker may be a divalent group other than a peptide linker, and may be a structural part that covalently links the HSA mutant and the protein (A). It is called “peptide linker”.
- peptide linker when the HSA mutant and the protein (A) are linked “through a peptide bond”, the case where they are directly linked by a peptide bond and the binding of both to a peptide linker It includes the case where it is connected by.
- the compound “HSA mutant-linked protein (A)” is referred to as “HSA mutation.
- body fusion protein (A) is also referred to as “body fusion protein (A)”.
- the linker is preferably 1 to 50, more preferably 1 to 17, more preferably 1 to 10, More preferably, it is composed of 1 to 6 amino acid residues, for example, 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31 or 25 to 29. For example, it is composed of only one amino acid residue or composed of 2, 3, 5, 6, or 20 amino acid residues.
- the amino acid residues constituting the peptide linker The group or amino acid sequence is not limited, but is preferably composed of glycine and serine.
- Preferred examples of peptide linkers include Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5), Examples include those comprising Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6) and those comprising these amino acid sequences. Any one of these amino acid sequences having a sequence of 2 to 10 times or 2 to 5 times in succession can be suitably used as a peptide linker, and any two or more of these amino acid sequences can be used.
- amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5) is 3 after the amino acid sequence Gly-Ser. Examples include those comprising a total of 20 amino acid sequences.
- an expression vector in which a DNA in which a gene encoding the other polypeptide is bound in-frame is prepared downstream of the gene encoding one polypeptide.
- a method of expressing a recombinant fusion protein by culturing a host cell transformed with this expression vector can be used in the present invention.
- the polypeptide comprising the amino acid sequence of the protein (A) comprises the amino acid sequence of the HSA mutant.
- a fusion protein in a form linked to either the N-terminus or C-terminus of the polypeptide is obtained.
- polypeptide comprising the amino acid sequence of the protein (A) is linked to the N-terminal side of the polypeptide comprising the amino acid sequence of the HSA variant, the polypeptide comprising the amino acid sequence of the protein (A) is encoded.
- An expression vector incorporating a DNA in which a gene encoding a polypeptide comprising the amino acid sequence of an HSA mutant is linked in-frame downstream of the gene to be used is used.
- a DNA sequence encoding the linker is inserted in frame between the genes encoding the two polypeptides.
- an expression vector incorporating a DNA encoding either of them is introduced into the host cell.
- the host cell that can be used for this purpose is not particularly limited as long as it can express the HSA mutant or HSA mutant fusion protein (A) of the present invention by introducing such an expression vector.
- Any of eukaryotic cells such as mammalian cells, yeast, plant cells, and insect cells, and prokaryotic cells such as Escherichia coli and Bacillus subtilis, but mammalian cells are particularly preferred.
- the host cell to be expressed as a sugar chain-modified protein (A) is selected from eukaryotic cells such as mammalian cells, yeast, plant cells, and insect cells.
- the Asn residue of the sequence part represented by Asn-Tyr-Thr which is usually generated by the 320th amino acid residue of wild-type HSA becoming threonine, or Asn-X-Thr ("X" is an amino acid other than proline)
- the Asn residue in the sequence represented by (residues) is N-linked glycosidated by allowing the HSA mutant fusion protein (A) to be expressed in eukaryotic cells.
- the type of the mammalian cell is not particularly limited, but a cell derived from human, mouse, or Chinese hamster is preferable, and a CHO cell derived from Chinese hamster ovary cell or mouse myeloma is particularly preferable.
- NS / 0 cells derived from are preferred.
- an expression vector used to incorporate and express a DNA fragment encoding the HSA mutant or HSA mutant fusion protein (A) of the present invention causes expression of the gene when introduced into a mammalian cell. If it can be used, there is no particular limitation.
- the gene incorporated in the expression vector is arranged downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells.
- a DNA sequence gene expression control site
- Examples of gene expression control sites that can be used in the present invention include a cytomegalovirus-derived promoter, SV40 early promoter, human elongation factor-1 ⁇ (EF-1 ⁇ ) promoter, human ubiquitin C promoter, and the like.
- Mammalian cells introduced with such an expression vector express the protein incorporated in the expression vector, but the expression level varies depending on the individual cells and is not uniform. Therefore, in order to efficiently produce the HSA mutant or HSA mutant fusion protein (A) of the present invention, a step of selecting cells having high expression levels from mammalian cells into which an expression vector has been introduced is necessary. It becomes. In order to perform this selection step, a gene that functions as a selection marker is incorporated into the expression vector.
- the most common selection marker is an enzyme (drug resistance marker) that degrades drugs such as puromycin and neomycin.
- Mammalian cells usually die in the presence of certain agents above a certain concentration.
- mammalian cells into which an expression vector incorporating a drug resistance marker gene has been introduced can degrade the drug with the expressed drug resistance marker and detoxify or attenuate it. It can survive even under.
- an expression vector incorporating a drug resistance marker as a selection marker is introduced into a mammalian cell, and the culture is continued while gradually increasing the concentration of the drug in a selective medium containing a drug corresponding to the drug resistance marker , Cells that can proliferate even in the presence of higher concentrations of drug are obtained. In the cells selected in this way, the expression level of the gene encoding the target protein incorporated in the expression vector is generally increased together with the drug resistance marker, and as a result, cells having a high expression level of the protein are selected.
- glutamine synthetase can be used as a selection marker.
- Glutamine synthase is an enzyme that synthesizes glutamine from glutamic acid and ammonia.
- an inhibitor of glutamine synthase such as methionine sulphoximine (MSX)
- MSX methionine sulphoximine
- the cells usually die.
- an expression vector incorporating glutamine synthetase as a selectable marker is introduced into a mammalian cell, the expression level of glutamine synthetase increases in the cell, so that it can grow even in the presence of a higher concentration of MSX. It becomes.
- the culture is continued while gradually increasing the concentration of MSX, cells that can proliferate even in the presence of a higher concentration of MSX can be obtained.
- the expression level of the gene encoding the target protein incorporated in the expression vector is generally increased together with glutamine synthase, and as a result, cells having a high expression level of the protein are selected. .
- DHFR dihydrofolate reductase
- mammalian cells into which an expression vector has been introduced are cultured in a selective medium containing a DHFR inhibitor such as methotrexate or aminopterin. If the culture is continued while gradually increasing the concentration of the DHFR inhibitor, cells that can proliferate even in the presence of a higher concentration of the DHFR inhibitor can be obtained. In the cells selected in this manner, the expression level of the gene encoding the target protein incorporated in the expression vector is generally increased together with DHFR, and as a result, cells having a high expression level of the protein are selected.
- a DHFR inhibitor such as methotrexate or aminopterin.
- G glutamine synthetase
- IVS internal ribosome binding site
- an expression vector for expressing a protein of interest comprising a first gene expression control site, a gene encoding the protein downstream thereof, an internal ribosome binding site downstream, and a glutamine synthesis further downstream
- An expression vector comprising a gene encoding an enzyme and further comprising a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression control site or a second gene expression control site different from the first gene expression control site can be suitably used for the production of the HSA mutant or HSA mutant fusion protein (A) of the present invention.
- the first gene expression control site or the second gene expression control site includes a cytomegalovirus-derived promoter, SV40 early promoter, human elongation factor-1 ⁇ promoter (hEF-1 ⁇ promoter), human ubiquitin C A promoter is preferably used, but the hEF-1 ⁇ promoter is particularly preferred.
- Internal ribosome binding sites include: Picornaviridae virus, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine intestinal virus, Siler's murine encephalomyelitis virus, Coxsackie B virus Preferred are those derived from the genome of a virus selected from the group consisting of 5 ′ untranslated region of a gene selected from the group consisting of: a human immunoglobulin heavy chain binding protein gene, a Drosophila antennapedia gene, and a Drosophila ultravitrax gene
- an internal ribosome binding site derived from the 5 ′ untranslated region of the mouse encephalomyocarditis virus genome is particularly preferred.
- the drug resistance gene suitably used is preferably a puromycin or neomycin resistance gene, and more preferably a puromycin resistance gene.
- an expression vector for expressing a target protein which is a human elongation factor-1 ⁇ promoter, a gene encoding the protein downstream thereof, and a 5 ′ untranslated region of the mouse encephalomyocarditis virus genome downstream.
- An expression vector comprising an internal ribosome binding site derived from, and a gene encoding glutamine synthetase further downstream, and further comprising another gene expression control site and a dihydrofolate reductase gene downstream thereof, the internal ribosome binding
- An expression vector in which a part of a plurality of initiation codons contained in a wild-type internal ribosome binding site is disrupted is the production of the HSA mutant or HSA mutant fusion protein (A) of the present invention.
- HSA mutant or HSA mutant fusion protein (A) of the present invention can be suitably used. Examples of such an expression vector include the expression vector described in WO2013 / 161958.
- an expression vector for expressing a target protein which is a human elongation factor-1 ⁇ promoter, a gene encoding the protein downstream thereof, and a 5 ′ untranslated region of the mouse encephalomyocarditis virus genome downstream.
- An expression vector comprising an internal ribosome binding site derived from, and a gene encoding glutamine synthetase further downstream, and further comprising another gene expression control site and a drug resistance gene downstream thereof, the internal ribosome binding site
- an expression vector in which a part of a plurality of initiation codons contained in the wild-type internal ribosome binding site is disrupted is used for the production of the HSA mutant or HSA mutant fusion protein (A) of the present invention. It can be used suitably.
- Examples of such expression vectors include pE-mIRES-GS-puro described in WO2012 / 063799 and pE-mIRES-GS-mNeo described in WO2013 / 161958.
- start codons There are three start codons (ATG) at the 3 'end of the internal ribosome binding site derived from the 5' untranslated region of the wild-type mouse encephalomyocarditis virus genome, and the sequence includes these three start codons.
- the portion is shown in SEQ ID NO: 7 (5′-ATGataatATGgccacaaccATG-3 ′: ATG of the disclosed codon is shown in capital letters).
- SEQ ID NO: 8 5'-atgataagcttgccacaaccatg-3 '
- pE-mIRES-GS-puro and pE-mIRES-GS-mNeo is an expression vector having an IRES containing the sequence represented by SEQ ID NO: 8.
- mammalian cells into which an expression vector incorporating a DNA fragment encoding the HSA mutant or HSA mutant linking protein (A) of the present invention has been introduced are selected in order to select cells with high expression levels. , Selective culture in selective medium.
- the concentration of the DHFR inhibitor contained in the selective medium is increased stepwise.
- the maximum concentration is preferably 0.25 to 5 ⁇ M, more preferably 0.5 to 1.5 ⁇ M, and even more preferably about 1.0 ⁇ M when the DHFR inhibitor is methotrexate.
- the concentration of the GS inhibitor contained in the selection medium is increased stepwise.
- the maximum concentration is preferably 100 to 1000 ⁇ M, more preferably 200 to 500 ⁇ M, and still more preferably about 300 ⁇ M.
- a medium not containing glutamine is generally used as the selective medium.
- the maximum concentration of puromycin contained in the selection medium is preferably 3 to 30 ⁇ g / mL, more preferably 5 to 20 ⁇ g / mL, and even more preferably. About 10 ⁇ g / mL.
- the maximum concentration of G418 contained in the selection medium is preferably 0.1 mg to 2 mg / mL, more preferably 0.5 to 1.5 mg / mL. , More preferably about 1 mg / mL.
- both the medium used for selective culture and the medium used for producing the recombinant protein described later are cultured and grown.
- Any medium can be used without particular limitation, but a serum-free medium is preferably used. Since HSA has the property of adsorbing components contained in serum, when HSA is produced using a medium containing serum, HSA adsorbing impurities in serum can be obtained. It is necessary to remove this impurity.
- the HSA mutant or HSA mutant fusion protein (A) of the present invention is particularly obtained by culturing cells expressing these in a serum-free medium.
- a serum-free medium By using a serum-free medium, the amount of impurities adsorbed on HSA can be reduced, so that the subsequent purification step can be simplified.
- recombinant protein producing cells Cells with high expression level of recombinant protein selected by selective culture are used for production of recombinant protein (recombinant protein producing cells). Production of recombinant protein is carried out by culturing recombinant protein-producing cells in a recombinant protein production medium. This culture is called production culture.
- the serum-free medium used as a recombinant protein production medium is, for example, 3 to 700 mg / L of amino acids, 0.001 to 50 mg / L of vitamins, and 0.3 to 10 g / L of monosaccharides.
- 0.1 to 10,000 mg / L of inorganic salt 0.001 to 0.1 mg / L of trace elements, 0.1 to 50 mg / L of nucleoside, 0.001 to 10 mg / L of fatty acid, and 0.01 to biotin ⁇ 1mg / L, hydrocortisone 0.1 ⁇ 20 ⁇ g / L, insulin 0.1 ⁇ 20mg / L, vitamin B12 0.1 ⁇ 10mg / L, putrescine 0.01 ⁇ 1mg / L, sodium pyruvate
- a medium containing 10 to 500 mg / L and a water-soluble iron compound is preferably used. If desired, thymidine, hypoxanthine, conventional pH indicators and antibiotics may be added to the medium.
- a DMEM / F12 medium (mixed medium of DMEM and F12) may be used as a basic medium, and each of these mediums is well known to those skilled in the art.
- the serum-free medium contains sodium hydrogen carbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, iron (II) sulfate, asparagine, aspartic acid, serine and polyvinyl alcohol.
- HG HAM modified (R5) medium may be used.
- serum-free media such as CD OptiCHO TM media, CHO-S-SFM II media or CD CHO media (Thermo Fisher Scientific, formerly Life Technologies), IS cho-V TM media (Irvine Scientific), EX-CELL TM 302 medium or EX-CELL TM 325-PF medium (SAFC Biosciences) can also be used as a basic medium.
- non-peptide linkers include polyethylene glycol (PEG), polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers , Lipid polymers, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof can be used.
- the peptide linker is a peptide chain composed of 1 to 50 amino acids linked by peptide or a derivative thereof, and the N-terminus and C-terminus thereof are either the HSA variant of the present invention or the desired protein, respectively.
- the HSA variant of the present invention and a desired protein are linked.
- HSA mutant PEG-linked protein (A) When the HSA mutant of the present invention is linked to the protein (A) using PEG as a non-peptide linker, when specifically mentioned, it is referred to as an HSA mutant PEG-linked protein (A).
- the HSA mutant PEG-linked protein (A) is obtained by binding the HSA mutant and PEG (PEGylated HSA mutant) and binding the protein (A) thereto, or by previously binding the protein (A) and PEG. (PEGylated bioactive protein (A)) and HSA mutants can be bound thereto.
- PEG modified with a functional group such as carbonate, carbonylimidazole, active ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde Is used.
- the functional group introduced into these PEGs mainly reacts with the amino group of the HSA mutant and protein (A) of the present invention, whereby the HSA mutant and protein (A) of the present invention are covalently bonded.
- PEG having an average molecular weight of about 300, about 500, about 1000, about 2000, about 4000, about 10,000, about 20000, etc. can be suitably used as a non-peptide linker.
- a PEGylated HSA variant comprises a HSA variant of the present invention and polyethylene glycol (ALD-PEG-ALD) having an aldehyde group as a functional group, wherein the molar ratio of HSA / (ALD-PEG-ALD) is 11, mixture so that such 12.5,15,110,120, this can be obtained by reacting by adding a reducing agent such as NaCNBH 3.
- a reducing agent such as NaCNBH 3
- the PEGylated HSA mutant is reacted with the protein (A) in the presence of a reducing agent such as NaCNBH 3 to obtain an HSA mutant PEG-linked protein.
- the HSA of the present invention can also be obtained by first binding the protein (A) and ALD-PEG-ALD to produce the PEGylated protein (A) and binding this to the HSA mutant of the present invention.
- a mutant PEG-linked protein (A) can be obtained.
- the protein (A) to be linked to the HSA mutant of the present invention is preferably a protein that exhibits physiological activity when administered to a living body, and is selected as desired.
- proteins include ⁇ -L-iduronidase, iduronic acid-2-sulfatase, glucocerebrosidase, ⁇ -galactosidase, GM2 activated protein, ⁇ -hexosaminidase A, ⁇ -hexosaminidase B, N -Acetylglucosamine-1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, galactosylceramidase, saposin C, allylsulfatase A, ⁇ -L-fucosidase, aspartylglucosaminidase, ⁇ -N-acetylgalactosaminidase, acid sphingomye eri Nase, ⁇ -galactos
- the protein (A) to be linked to the HSA mutant is not particularly limited to the species from which it is derived, but is preferably a mammal-derived protein, more preferably human, African green monkey, etc. Proteins derived from rodents such as primates, mice, rats, and Chinese hamsters, rabbits, and dogs, particularly preferably human-derived proteins.
- the protein (A) is not limited to the wild type. That is, a mutant in which one or more amino acids are substituted, deleted, and / or added to the wild-type amino acid sequence, and retains the physiological activity of the original protein (A), It may be one that functions by antagonizing the wild-type protein (A) (this affects the function of the endogenous protein (A)).
- the number of amino acids that may be substituted, deleted, and / or added is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3 for each mutation type. is there. These amino acid substitutions, deletions and / or additions can occur in combination.
- the HSA mutant-linked protein (A) of the present invention has a higher stability in blood and a longer half-life than the original protein (A) to which the HSA mutant is not linked.
- the blood half-life (t 1/2 ⁇ ) when administered to cynomolgus monkeys by subcutaneous injection is approximately 5 hours or more, and is extremely stable in blood.
- the blood half-life (t 1/2 ⁇ ) of the HSA mutant-linked human growth hormone of the present invention is the half-life in blood when a single subcutaneous administration is made to male cynomolgus monkeys at a dose of 0.5 to 10 mg / kg. (T 1/2 ⁇ ) is 5 to 40 hours.
- the HSA variant-linked protein (A) of the present invention can be used as a pharmaceutical due to the activity exhibited by the protein (A) portion when administered to a living body.
- the “living body” refers to mammals including humans, particularly preferably human living bodies.
- the HSA variant linking protein (A) of the present invention has improved stability in blood, it has been unstable in blood and has been degraded immediately after administration and has not been able to show sufficient efficacy. Even the protein (A) can be stabilized in blood and exert its physiological activity by linking the HSA variant of the present invention to the protein (A), which opens the way to development as a medicine.
- the protein (A) that can be conventionally used as a pharmaceutical agent is linked to the HSA variant of the present invention, thereby further improving the stability in blood and having a long-term activity. Since it can be kept in the blood in the held state, the administration frequency or dose of the protein (A) can be reduced.
- the administration frequency of a drug that needs to be administered every day can be linked to the HSA variant of the present invention, for example, every 3 to 30 days.
- the dose of the drug can be reduced to, for example, 1/3 to 1/100 in molar ratio.
- a medicament comprising the HSA variant linking protein (A) of the present invention as an active ingredient can be administered as an injection intravenously, intramuscularly, intraperitoneally or subcutaneously.
- the route of administration of the drug is appropriately selected depending on the dosage form, application, etc.
- These injections can be supplied as lyophilized formulations or aqueous solutions.
- an aqueous liquid preparation When an aqueous liquid preparation is used, it may be in a form filled in a vial or may be supplied as a prefilled preparation that is pre-filled in a syringe. In the case of lyophilized preparations, dissolve and restore in aqueous media before use.
- human growth hormone is a protein composed of 191 amino acids having the amino acid sequence represented by SEQ ID NO: 9.
- human growth hormone or hGH
- human growth hormone or hGH
- 22K human growth hormone or 22KhGH
- 22KhGH in addition to wild-type 22KhGH having the amino acid sequence shown by SEQ ID NO: 9, one or more amino acids are substituted.
- 22KhGH mutants that have been deleted and / or added and that have growth promoting activity.
- the number of amino acids that may be substituted, deleted and / or added is preferably 1 to 8, more preferably 1 to 4, and still more preferably 1 to 2 for each mutation type.
- Wild-type 20K human growth hormone is a deletion of 15 amino acids from the 32nd to 46th positions counted from the N-terminal out of 191 amino acids constituting wild-type 22K growth hormone (SEQ ID NO: 9). It is a protein having a growth promoting activity consisting of an amino acid sequence composed of 176 amino acids (SEQ ID NO: 10).
- 20K human growth hormone or 20KhGH
- 20KhGH one or more amino acids are substituted for the sequence.
- Mutants of 20 KhGH corresponding to those deleted and / or added and those having growth promoting activity are also included.
- the number of amino acids that may be substituted, deleted and / or added is preferably 1 to 8, more preferably 1 to 4, and still more preferably 1 to 2 for each mutation type.
- a preparation (hGH preparation) containing hGH with a molecular weight of about 22 KD as an active ingredient, produced as a recombinant protein using Escherichia coli introduced with the hGH gene, is a growth hormone secretory short stature, short stature in Turner syndrome, bone Widely used as a therapeutic agent for SGA short stature without end-line closure, short stature due to chronic renal failure, short stature in Prader-Willi syndrome, and short stature in cartilage dystrophy.
- the hGH preparation is administered subcutaneously or intramuscularly, whereby the component hGH circulates in the blood and exhibits the effect of promoting the growth of the patient by its growth promoting activity.
- hGH preparations are widely used clinically as therapeutic agents for adult growth hormone secretion deficiency.
- abnormal lipid metabolism is observed, but administration of the hGH preparation normalizes the patient's lipid metabolism and improves the patient's QOL.
- Growth hormone has also been applied clinically as a therapeutic agent for AIDS exhaustion.
- Examples of hGH preparations such as growth hormone deficiency short stature and adult growth hormone secretion deficiency include GLOJECT (registered trademark).
- a protein that employs human growth hormone as the protein (A) that is a linking partner of human serum albumin variant (mHSA) is referred to as “human serum albumin variant-linked human growth hormone” or “mHSA-linked hGH”, etc.
- human serum albumin mutant fusion human growth hormone when linked via a peptide bond, it is also referred to as “human serum albumin mutant fusion human growth hormone”, “mHSA fusion hGH” or the like.
- a method of expressing a recombinant protein by preparing an expression vector incorporating a DNA fragment to which a gene encoding a polypeptide of this gene is linked and culturing host cells transformed with this expression vector is generally used. And can be used in the present invention.
- the polypeptide containing the amino acid sequence of hGH is the N-terminal side or C-terminal of the polypeptide containing the amino acid sequence of the HSA variant of the present invention. Linked to either side directly or indirectly through a linker.
- the gene encoding the polypeptide containing the amino acid sequence of hGH is downstream of the gene encoding the polypeptide of the present invention.
- An expression vector incorporating a DNA fragment in which a gene encoding a polypeptide containing the amino acid sequence of an HSA variant is linked in frame is used.
- a DNA sequence encoding the linker is placed in frame between the genes encoding the two polypeptides.
- the gene encoding the polypeptide containing the amino acid sequence of hGH is upstream of the gene encoding the polypeptide of the present invention.
- An expression vector incorporating a DNA fragment in which a gene encoding a polypeptide containing the amino acid sequence of an HSA variant is linked in frame is used.
- a DNA sequence encoding the linker is placed in frame between the genes encoding the two polypeptides.
- polypeptides are prepared separately as recombinant proteins, and then There is a method of connecting two via a non-peptide linker or a peptide linker.
- Non-peptide linkers include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextrans, polyvinyl ethers, biodegradable polymers, lipid polymers, Chitins and hyaluronic acid, derivatives thereof, or combinations thereof can be used.
- the peptide linker is a peptide chain composed of 1 to 50 amino acids linked by peptide or a derivative thereof, and the N-terminus and C-terminus thereof are either the HSA variant of the present invention or the desired protein, respectively. By forming a peptide bond, the HSA variant of the present invention and a desired protein are linked.
- HSA mutant PEG-linked protein (A) When the HSA mutant and protein (A) are linked using PEG as a non-peptide linker, when the linker is specified, it is referred to as HSA mutant PEG-linked protein (A). That is, when hGH is selected as the protein (A), it is referred to as HSA mutant PEG-linked hGH.
- the HSA mutant PEG-linked hGH first binds the HSA mutant and PEG (PEGylated HSA mutant) and then binds hGH to this, or first binds hGH and PEG (PEGylated hGH). ), And can be produced by binding an HSA variant thereto.
- PEG modified with a functional group such as carbonate, carbonylimidazole, active ester of carboxylic acid, azlactone, cyclic imidothione, isocyanate, isothiocyanate, imidate, or aldehyde Is used.
- the functional group introduced into these PEGs mainly reacts with the HSA variant of the present invention and the amino group of the hGH molecule, whereby the HSA variant of the present invention and hGH are covalently bonded.
- MW average molecular weight
- PEG having an average molecular weight of about 300, about 500, about 1000, about 2000, about 4000, about 10,000, about 20000, etc. can be suitably used as a non-peptide linker.
- a PEGylated HSA variant comprises a HSA variant of the present invention and polyethylene glycol (ALD-PEG-ALD) having an aldehyde group as a functional group, wherein the molar ratio of HSA / (ALD-PEG-ALD) is 11, It is obtained by mixing the mixture so that it becomes 12.5, 15, 110, 120, etc., and adding a reducing agent such as NaCNBH 3 to this. The PEGylated HSA mutant is then reacted with hGH in the presence of a reducing agent such as NaCNBH 3 to obtain an HSA mutant PEG-linked hGH.
- the HSA mutant PEG-linked hGH of the present invention can also be obtained by previously combining hGH and ALD-PEG-ALD to prepare PEGylated hGH and binding this to the HSA mutant. it can.
- the amino acid sequence represented by SEQ ID NO: 9 is not linked to the N-terminal of HSA (A320T) having the amino acid sequence represented by SEQ ID NO: 3 via a linker.
- HSA (A320T) having the amino acid sequence represented by SEQ ID NO: 11
- 22KhGH linked in this order are referred to as “22K human growth hormone-mHSA” or “22KhGH-mHSA”.
- mHSA-22K human growth hormone a peptide in which the N-terminus of 22K human growth hormone is bonded to the C-terminus of HSA (A320T) via a peptide bond without using a linker is referred to as “mHSA-22K human growth hormone” or “mHSA-22KhGH”.
- the C-terminus of 20K human growth hormone having the amino acid sequence represented by SEQ ID NO: 10 is bound to the N-terminus of human serum albumin (A320T) having the amino acid sequence represented by SEQ ID NO: 3 by a peptide bond without using a linker.
- A320T human serum albumin
- the mHSA-linked hGH having the amino acid sequence represented by SEQ ID NO: 12 is referred to as “20K human growth hormone-mHSA” or “20KhGH-mHSA”.
- mHSA-20K human growth hormone a substance in which the N-terminus of 20K human growth hormone is linked to the C-terminus of human serum albumin (A320T) via a peptide bond without using a linker is referred to as “mHSA-20K human growth hormone” or “mHSA-22KhGH”.
- the HSA mutant-linked human growth hormone of the present invention is characterized in that the blood half-life (t 1/2 ⁇ ) when administered to cynomolgus monkeys by subcutaneous injection is approximately 10 hours or more and is extremely stable in blood. .
- the HSA mutant-linked human growth hormone of the present invention can be used as a medicine. It can also be used as a medicine by cooperating the functions of human growth hormone and HSA mutant in vivo.
- the HSA mutant-linked human growth hormone of the present invention is extremely stable in blood. Therefore, according to the present invention, human growth hormone can be stabilized in the blood and can remain in the blood in a state where the activity is maintained for a long time. It is possible to reduce the dose. For example, by linking the administration frequency of a drug that needs to be administered daily with the HSA variant of the present invention, it is possible to administer the drug every 3 to 30 days, for example. In addition, the dose of the drug can be reduced to 1/3 to 1/100 in molar ratio.
- the HSA mutant-linked human growth hormone of the present invention has growth hormone deficiency short stature, short stature in Turner syndrome, short stature due to chronic renal failure, short stature in Praderwilly syndrome, short stature in cartilage dystrophy.
- SGA short stature, all without epiphyseal closure, adult growth hormone secretion deficiency, exhaustion due to AIDS, and exhaustion due to anorexia can be used as a target drug
- the symptoms can be improved by applying growth-promoting activities such as promotion of cartilage formation, promotion of protein assimilation, and other physiological activities such as body composition and lipid metabolism-improving action. It can be used as a therapeutic agent for diseases.
- the preferred dose per dose is 0.01 to 0.7 mg / Kg body weight.
- the preferred dose per dose is 0.015-1.4 mg / Kg body weight.
- the preferred dose per dose is 0.01 to 1.4 mg / Kg body weight.
- the preferred dose per dose is 0.012-0.98 mg / Kg body weight.
- the preferred dose per dose is 0.015-1.4 mg / Kg body weight.
- the preferred dose per dose is 0.012-1.9 mg / Kg body weight.
- the preferred dose per dose is 0.001 to 0.34 mg / Kg body weight.
- the preferred dose per dose is 0.005 to 0.4 mg / Kg body weight.
- the dose should be changed appropriately according to the patient's laboratory findings.
- the preferred administration interval of mHSA-22KhGH in these diseases is once every 7 to 30 days, and once every 7 to 14 days, every 10 to 20 days, or once every 14 to 21 days, depending on the patient's examination findings, etc. And should be changed as appropriate.
- the administration method is preferably subcutaneous injection, intramuscular injection or intravenous injection, and more preferably subcutaneous injection or intramuscular injection.
- a medicament comprising the HSA variant linking protein of the present invention as an active ingredient can be administered as an injection, intravenously, intramuscularly, intraperitoneally, subcutaneously or intraventricularly.
- injections can be supplied as lyophilized formulations or aqueous solutions.
- an aqueous liquid preparation When an aqueous liquid preparation is used, it may be in a form filled in a vial or may be supplied as a prefilled preparation that is pre-filled in a syringe. In the case of lyophilized preparations, dissolve and reconstitute in an aqueous medium before use.
- the pE-neo vector was digested with restriction enzymes (SfiI and BstXI), and an approximately 1 kbp region containing a neomycin resistance gene was excised (FIG. 2).
- the hygromycin gene was amplified by PCR using pcDNA3.1 / Hygro (+) (Invitrogen) as a template and using the primer Hyg-Sfi5 ′ (SEQ ID NO: 13) and the primer Hyg-BstX3 ′ (SEQ ID NO: 14) ( Figure 2).
- the amplified hygromycin gene was digested with restriction enzymes (SfiI and BstXI) and inserted into the above pE-neo vector to construct a pE-hygr vector (FIG. 2).
- the expression vector pPGKIH (Miyahara M. et.al., J. Biol. Chem. 275,613-618 (2000)) is digested with restriction enzymes (XhoI and BamHI), and the internal ribosome derived from mouse encephalomyocarditis virus (EMCV) Nucleotide sequence IRES-Hygr-mPGKpA (SEQ ID NO: 15: 5 ′ end to base) including binding site (IRES), hygromycin resistance gene (Hygr gene) and mouse phosphoglycerate kinase (mPGK) polyadenylation region (mPGKpA)
- the region consisting of 1 to 6 is the “XhoI site”, the bases 120 to 715 followed by the bases 716 to 718 (atg) are the “internal ribosome binding derived from the 5 ′ untranslated region of the mouse encephalomyocarditis virus genome "A base sequence including a site”, a
- DNA fragment containing a part of EMCV IRES was amplified by PCR using pBSK (IRES-Hygr-mPGKpA) as a template and using primer IRES5 ′ (SEQ ID NO: 17) and primer IRES3 ′ (SEQ ID NO: 18).
- This DNA fragment was digested with restriction enzymes (XhoI and HindIII) and inserted between the XhoI and HindIII sites of pBSK (IRES-Hygr-mPGKpA), and this was designated as pBSK (NotI-IRES-Hygr-mPGKpA) ( Fig. 3-2).
- pBSK NotI-IRES-Hygr-mPGKpA
- restriction enzymes NotI and BamHI
- mPGKP5 ′ SEQ ID NO: 19
- primer mPGKP3 ′ SEQ ID NO: 20
- a nucleotide sequence SEQ ID NO: 21: from the 5 ′ end
- Bases 4 to 9 are “BglII site”
- the subsequent region consisting of bases 10 to 516 is “base sequence including promoter region of mouse phosphoglycerate kinase gene”
- the subsequent region consisting of bases 524 to 529 is “ A DNA fragment consisting of “EcoRI site” was amplified.
- This DNA fragment was digested with restriction enzymes (BglII and EcoRI) and inserted between the BglII and EcoRI sites of pCI-neo (Promega), and this was designated as pPGK-neo (FIG. 3-4).
- pE-IRES-Hygr is digested with restriction enzymes (NotI and BamHI) to cut out a DNA fragment (IRES-Hygr) and inserted between NotI and BamHI sites of pPGK-neo, which is called pPGK-IRES-Hygr. (Fig. 3-5).
- CDNA was prepared from CHO-K1 cells, and using this cDNA as a template, a DNA fragment containing the GS gene was amplified by PCR using primer GS5 ′ (SEQ ID NO: 22) and primer GS3 ′ (SEQ ID NO: 23). This DNA fragment was digested with restriction enzymes (BalI and BamHI) and inserted between the BalI and BamHI sites of pPGK-IRES-Hygr to obtain pPGK-IRES-GS- ⁇ polyA (FIGS. 3-6).
- restriction enzymes BalI and BamHI
- This DNA fragment was digested with restriction enzymes (AflII and BstXI) and inserted between the AflII and BstXI sites of the expression vector pE-neo, which was designated as pE-puro (FIGS. 3-7).
- DNA fragment containing SV40 late polyadenylation region was amplified by PCR using pE-puro as a template and primer SV40polyA5 ′ (SEQ ID NO: 28) and primer SV40polyA3 ′ (SEQ ID NO: 29).
- This DNA fragment was digested with restriction enzymes (NotI and HpaI) and inserted between the NotI and HpaI sites of the expression vector pE-puro to obtain pE-puro (XhoI) (FIGS. 3-8).
- pPGK-IRES-GS- ⁇ polyA is digested with restriction enzymes (NotI and XhoI), a DNA fragment containing the IRES-GS region is excised, and this DNA fragment is located between the NotI and XhoI sites of the expression vector pE-puro (XhoI). This was used as pE-IRES-GS-puro ( Figure 3-9).
- the region from EMCV IRES to GS is obtained by PCR.
- the amplified DNA fragment was amplified by mutating the start codon (atg) located second from the 5 ′ side of the EMCV IRES.
- a DNA fragment containing the above-mentioned region from IRES to GS was amplified by PCR.
- This DNA fragment is digested with restriction enzymes (NotI and PstI), and the excised DNA fragment is inserted between the NotI and PstI sites of the expression vector pE-IRES-GS-puro.
- pE-mIRES-GS-puro was used (FIG. 4).
- Example 2 Construction of HSA-22KhGH expression vector
- the amino acid sequence of the fusion protein HSA-22KhGH obtained by fusing the C-terminus of wild-type HSA (SEQ ID NO: 1) and the N-terminus of 22KhGH is represented by SEQ ID NO: 32. Shown in In this amino acid sequence, the 1st to 585th amino acid residues correspond to the amino acid sequence of wild type mature HSA (SEQ ID NO: 1), and the 586th to 776th amino acid residues correspond to the 22KhGH amino acid sequence.
- a DNA having the base sequence shown in SEQ ID NO: 33 containing a gene encoding HSA-22KhGH (HSA-22KhGH gene) was chemically synthesized.
- bases 11 to 82 encode the leader peptide of HSA
- bases 83 to 1837 encode mature HSA
- bases 1838 to 2410 encode mature hGH.
- This DNA was digested with restriction enzymes (MluI and NotI) and inserted between the MluI and NotI sites of pE-mIRES-GS-puro prepared in Example 1 to express the vector pE-mIRES- for expression of HSA-22KhGH.
- GS-puro HSA-22KhGH
- Example 3 Construction of mHSA-22KhGH expression vector Fusion protein having the amino acid sequence shown in SEQ ID NO: 34, which is a fusion of the C-terminal of HSA (A320T) (SEQ ID NO: 3) and the N-terminal of 22KhGH was designated mHSA-22KhGH.
- SEQ ID NO: 34 the 1st to 585th amino acid residues correspond to the amino acid sequence of mHSA, and the 586th to 776th amino acid residues correspond to the amino acid sequence of 22KhGH.
- Example 4 Construction of 22KhGH-HSA expression vector Fusion protein having the amino acid sequence shown in SEQ ID NO: 37, wherein the C terminus of 22KhGH is fused with the N terminus of wild type HSA (SEQ ID NO: 1) was set to 22KhGH-HSA.
- SEQ ID NO: 37 the 1st to 191st amino acid residues correspond to the 22KhGH amino acid sequence, and the 192th to 776th amino acid residues correspond to the amino acid sequence of HSA.
- a DNA having the base sequence shown in SEQ ID NO: 38 containing a gene encoding 22KhGH-HSA (22KhGH-HSA gene) was chemically synthesized.
- bases 11 to 88 encode the leader peptide of hGH
- bases 89 to 661 encode mature hGH
- bases 662 to 2416 encode mature HSA.
- This DNA is digested with restriction enzymes (MluI and NotI) and inserted between the MluI site and NotI site of pE-mIRES-GS-puro prepared in Example 1 to obtain pE-, a vector for expressing HSA-22KhGH.
- mIRES-GS-puro 22KhGH-HSA
- Example 5 Construction of 22KhGH-mHSA expression vector Fusion having 22 KhGH C-terminal and HSA (A320T) (SEQ ID NO: 3) N-terminal fusion having the amino acid sequence shown in SEQ ID NO: 39
- the protein was 22KhGH-mHSA.
- a DNA fragment containing was amplified. This DNA fragment was self-annealed to construct pE-mIRES-GS-puro (22KhGH-mHSA), which is a 22KhGH-mHSA expression vector.
- Example 6 Preparation of fusion protein-expressing cells
- Cells for expressing each fusion protein of HSA-22KhGH, mHSA-22KhGH, 22KhGH-HSA, and 22KhGH-mHSA were prepared as follows.
- HSA-22KhGH expression vector pE-mIRES prepared in Examples 2-5 using Gene Pulser Xcell electroporation system (Bio Rad) on CHO-K1 cells, which are cells derived from Chinese hamster ovary.
- HSA-22KhGH mHSA-22KhGH expression vector pE-mIRES-GS-puro
- mHSA-22KhGH mHSA-22KhGH expression vector pE-mIRES-GS-puro
- 22KhGH-HSA 22KhGH-HSA
- 22KhGH -An mHSA expression vector pE-mIRES-GS-puro 22KhGH-mHSA
- Cells into which the respective expression vectors have been introduced are selectively cultured using CD OptiCHO TM medium (Thermo Fisher Scientific) containing methionine sulfoximine (SIGMA) and puromycin (SIGMA), and HSA-22KhGH-expressing cells, mHSA-22KhGH-expressing cells, 22KhGH-HSA-expressing cells, and 22KhGH-mHSA-expressing cells were established, respectively.
- CD OptiCHO TM medium Thermo Fisher Scientific
- SIGMA methionine sulfoximine
- SIGMA puromycin
- HSA-22KhGH-expressing cells mHSA-22KhGH-expressing cells
- 22KhGH-HSA-expressing cells 22KhGH-mHSA-expressing cells
- 22KhGH-mHSA-expressing cells were established, respectively.
- concentrations of methionine sulphoximine and puromycin are increased stepwise, and finally the methionine sulphoximine concentration is 300 ⁇ M and the
- HSA-22KhGH expression cells, mHSA-22KhGH expression cells, 22KhGH-HSA, and 22KhGH-mHSA expression cells thus obtained are collectively referred to as HSA-hGH fusion protein-expressing cells, and these cells are cultured.
- the resulting fusion protein of HSA and hGH is collectively referred to as HSA-hGH fusion protein.
- Example 7 Culture of fusion protein-expressing cells HSA-22KhGH-expressing cells, mHSA-22KhGH-expressing cells, 22KhGH-HSA-expressing cells, and 22KhGH-mHSA-expressing cells were cultured as follows. Methionine sulfoximine and puromycin were added to CD OptiCHO TM medium (Thermo Fisher Scientific) to a concentration of 300 ⁇ M and 10 ⁇ g / mL, respectively, to prepare a cell culture medium. Each expression cell prepared in Example 6 was added to 5 mL of cell culture medium so that the cell density was 2 ⁇ 10 5 cells / mL, and cultured at 37 ° C. in the presence of 5% CO 2 . Every 5 days, the cells were transferred to a new culture medium so that the cell density was 2 ⁇ 10 5 cells / mL, and subculture was performed.
- CD OptiCHO TM medium Thermo Fisher Scientific
- Example 8 Purification of HSA-hGH fusion protein HSA-22KhGH, mHSA-22KhGH, 22KhGH-HSA, and 22KhGH-mHSA were purified as follows. Each expression cell subcultured in Example 7 was suspended in a cell culture medium at a density of 2 ⁇ 10 5 cells / mL so that the total amount was 240 mL. 30 mL of this cell suspension was added to 8 dishes each having a diameter of 15 cm and cultured at 37 ° C. in the presence of 5% CO 2 for 5 days. After completion of the culture, the medium was collected and filtered through a membrane filter (pore size 0.22 ⁇ m, Millipore) to obtain a culture supernatant. Subsequently, 1M HEPES (pH 8.0) was added to each culture supernatant to adjust the pH to 7.0 to 7.2.
- a membrane filter pore size 0.22 ⁇ m, Millipore
- Polypropylene column (Poripureppu TM column, Bio-Rad), the anti-human growth hormone antibodies coupled to form a resin (Capture Select TM anti hGH resin, Thermo Fisher Scientific , Inc.) in 5mL filled a column volumes 5 volumes The resin was equilibrated with 10 mM HEPES buffer (pH 7.5) containing 500 mM NaCl. Next, the culture supernatant adjusted for pH was loaded onto the column at a flow rate of about 2.5 mL / min, and the HSA-hGH fusion protein was adsorbed to the resin.
- each HSA-hGH fusion protein was eluted from the resin with 0.1 M glycine buffer (pH 3.0) containing 100 mM NaCl in 5 column volumes.
- the eluted fraction containing the HSA-hGH fusion protein was collected, and 7% (v / v) 1M HEPES buffer (pH 8.0) was immediately added.
- the concentration of the HSA-hGH fusion protein contained in the eluted fraction was measured using Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific) with BSA as a standard substance.
- This DNA fragment was used as a mega primer.
- PCR was performed using human lung-derived cDNA as a template and using primer K708 (SEQ ID NO: 43) and primer K709 (SEQ ID NO: 44) to amplify a DNA fragment containing the full length of the hGHR gene.
- the obtained PCR product was subjected to agarose electrophoresis and purified using QIAEX II.
- PCR was performed using the purified DNA fragment containing the full length of the hGHR gene as a template using the above megaprimer and primer K709 (SEQ ID NO: 44), and the full length of hGHR having the hGHR ECD artificially synthesized gene sequence on the 5 ′ side was determined.
- a DNA fragment having the base sequence shown in SEQ ID NO: 45 containing the encoding gene was amplified. This DNA fragment was digested with restriction enzymes (MluI and NotI) and incorporated between MluI and NotI of the retroviral vector pMX-II (Ono Y., Oncogene. 19. 3050-8 (2000)).
- the retroviral vector for hGHR expression (hGHR / pMX-II) was used.
- 293 cells (Dainippon Pharmaceutical Co., Ltd.) were suspended in DMEM medium containing 10 mL of 10% FBS, and this was added to a 10 cm dish for cell culture at 37 ° C. in the presence of 5% CO 2. Cultured for 24 hours.
- the 293 cells used here are human fetal kidney cells transformed with adenovirus E1 gene.
- Opti-MEMI TM medium (Thermo Fisher Scientific)
- 15 ⁇ L of X-tremeGENE 9 DNA Transfection Reagent (Roche) was added and mixed, and 5 ⁇ g of retroviral packaging vector pCL-Eco ( IMGENEX) and 5 ⁇ g of hGHR / pMX-II were added and mixed.
- the mixture was allowed to stand at room temperature for 15 minutes, and then added to a 10 cm dish in which the 293 cells were cultured for 24 hours.
- the cells were cultured in the presence of 5% CO 2 at 37 ° C. for 24 hours, and then the medium was centrifuged at 3000 rpm for 5 minutes to collect the supernatant.
- the collected supernatant was used as an hGHR-expressing retrovirus solution.
- WEHI-3 cells were cultured in 10% FBS-containing RPMI1640 medium, and the medium was centrifuged at 3000 rpm for 5 minutes to collect the supernatant.
- 500 ⁇ L of the culture supernatant of WEHI-3 cells and 2.5 mL of RPMI 1640 medium containing 10% FBS were added and mixed.
- This mixture was added to 2 ⁇ 10 6 BaF3 cells (RIKEN), which is an IL-3-dependent cell line, and the cells were suspended.
- This cell suspension was transferred to a 75 cm 2 culture flask, and the cells were cultured at 37 ° C.
- Example 10 Measurement of cell proliferation activity using BaF3 / hGHR cells The cell proliferation activity of the HSA-hGH fusion protein was evaluated using BaF3 / hGHR cells prepared by the method described in Example 9.
- BaF3 / hGHR cells in logarithmic growth phase were washed 3 times with PBS, diluted to 1 ⁇ 10 6 cells / mL with 15 mL of RPMI 1640 medium containing 1% horse serum, and 37% in the presence of 5% CO 2.
- the cells were cultured at 16 ° C for 16 hours. After the culture, the cells were diluted with the same medium to 3 ⁇ 10 5 cells / mL, and 100 ⁇ L was seeded in each well of a 96-well culture plate.
- the HSA-hGH fusion protein (HSA-22KhGH, mHSA-22KhGH, 22KhGH-HSA and 22KhGH-mHSA) purified in Example 8 was diluted, and each of the 7 concentrations ( 90.3 nM, 18.1 nM, 3.6 nM, 0.72 nM, 0.14 nM, 0.029 nM, and 0.0058 nM).
- the absorbance was measured at 490 nm on the vertical axis, and the molar concentration (nM) of each specimen was plotted on the horizontal axis, and the measured values were plotted. Since the absorbance at 490 nm indicates the relative value of the number of living cells, the curve obtained by plotting the measured value shows the correlation between the concentration of the specimen and the amount of cell proliferation, and is obtained from this curve. the maximum value of the cell proliferation amount, the concentration of the analyte when the growth of cells is 50% was calculated as EC 50. Note that the measurement was performed three times for each specimen.
- Example 11 Pharmacokinetics and drug efficacy analysis using cynomolgus monkeys
- HSA-hGH fusion proteins HSA-22KhGH, mHSA-22KhGH, 22KhGH-HSA and 22KhGH-mHSA
- the administration of HSA-22KhGH was performed using 3 cynomolgus monkeys, and the administration of mHSA-22KhGH, 22KhGH-HSA and 22KhGH-mHSA was performed using 1 cynomolgus monkey each.
- peripheral blood of the animals was collected 15 minutes after administration, 1, 4, 8, 12, 24, 48, 72, 120, 168, and 216 hours.
- the blood was collected in an EDTA2-potassium-containing blood collection tube, cooled on ice, and centrifuged (1700 ⁇ g, 5 minutes, 4 ° C.) to separate plasma.
- Cmax, AUC 0-216h , AUC 0-inf and t 1/2 ⁇ were determined by pharmacokinetic analysis.
- the efficacy of the HSA-hGH fusion protein was analyzed as follows using IGF-1 secretion promotion as an index.
- Peripheral blood was collected before administration of the HSA-hGH fusion protein, 6, 12 hours after administration, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days, and plasma was collected from the peripheral blood by the above procedure.
- the concentration of IGF-1 contained in plasma was measured by the method described in detail in Example 13, and the efficacy was analyzed by plotting the IGF-1 concentration on the vertical axis and the time after administration on the horizontal axis.
- another cynomolgus monkey was prepared as a control, and 22 KhGH (Gloject (registered trademark)) was administered subcutaneously at a dose of 0.3 mg / kg for 7 consecutive days. Concentration was measured similarly.
- Example 12 Quantification of HSA-hGH fusion protein in plasma
- the hybridoma cells obtained by fusing were obtained by culturing.
- the mouse anti-hGH monoclonal antibody was dialyzed against a 0.1 M NaHCO 3 solution (pH 9), and then the antibody concentration in the solution was measured using NanoDrop TM (Thermo Scientific).
- EZ-Link TM NHS-LC-Biotin (Thermo Fisher Scientific) dissolved in DMSO at a concentration of 5 mg / mL was added to the antibody solution at a ratio of 60 ⁇ g NHS-LC-Biotin per mg of antibody, and room temperature. Then, the reaction solution was dialyzed with PBS to obtain a biotinylated mouse anti-hGH monoclonal antibody.
- a mouse anti-HSA monoclonal antibody was used as a primary antibody
- a biotinylated mouse anti-hGH monoclonal antibody was used as a secondary antibody.
- the concentration of HSA-hGH fusion protein in plasma was measured by electrochemiluminescence (ECL) immunoassay using Sector Imager 6000 (Meso Scale Diagnostics).
- ECL electrochemiluminescence
- a secondary antibody labeled with the ruthenium complex SULFO-TAG is electrochemically stimulated on the plate, and light emission at a wavelength of 620 nm due to electrolytic oxidation / reduction of SULFO-TAG is detected with a CCD camera.
- This is a method for quantifying a specimen. Measurements were generally performed as follows according to the Sector Imager 6000 instruction manual.
- Mouse anti-HSA monoclonal antibody was added to High Bind Plate (Meso Scale Diagnostics) and allowed to stand for 1 hour to immobilize anti-HSA antibody (primary antibody) on the plate.
- Superblock Blocking buffer in PBS Thermo Fisher Scientific
- PBST PBS containing 0.05% Tween20
- the specimen was added and shaken for 1 hour.
- biotinylated mouse anti-hGH monoclonal antibody (secondary antibody) was added and shaken for 1 hour.
- Example 13 Quantification of IGF-1 in plasma IGF-1 contained in plasma was quantified by ELISA using Human IGF-I Quantikine ELISA kit (R & D systems).
- FIG. 5 shows the measured values with the absorbance at 490 nm on the vertical axis and the molar concentration (nM) of each specimen on the horizontal axis. It is a figure which shows the measurement result of the cell growth activity using the BaF3 / hGHR cell plotted. Table 1 shows the results of obtaining the EC 50 of each specimen from this figure.
- the EC 50 of HSA-22KhGH and mHSA-22KhGH which are 22KhGH linked to the C-terminal side of human serum albumin, is 1.38 ⁇ 10 ⁇ 1 nM and 1.53 ⁇ 10 respectively. a -1 nM, both have substantially the same cell proliferation activity.
- 22KhGH-HSA and 22KhGH-mHSA in which 22KhGH is linked to the N-terminal side of human serum albumin their EC 50 are 8.78 ⁇ 10 ⁇ 1 nM and 1.20 nM, respectively. These were also shown to have almost the same cell proliferation activity.
- FIG. 6 shows the concentration of HSA-hGH fusion proteins (HSA-22KhGH, mHSA-22KhGH, 22KhGH-HSA and 22KhGH-mHSA) in cynomolgus monkey serum on the vertical axis and HSA on the horizontal axis.
- FIG. 7 is a graph showing the results of pharmacokinetic analysis of HSA-hGH fusion protein, in which the elapsed time after administration of the -hGH fusion protein is plotted.
- Table 2 shows the results of calculating Cmax, AUC 0-216h , AUC 0-inf, and t 1/2 ⁇ for each specimen.
- AUC is 752 ⁇ 55 ⁇ g ⁇ hr / mL for AUC 0-inf of HSA-22KhGH and mHSA-22KhGH, which are 22KhGH linked to the C-terminal side of human serum albumin, A value of 737 ⁇ g ⁇ hr / mL is shown.
- AUC 0-inf of 22KhGH-HSA and 22KhGH-mHSA in which 22KhGH is linked to the N-terminal side of human serum albumin is 2220 ⁇ g ⁇ hr / mL and 3260 ⁇ g ⁇ hr / mL, respectively.
- these results show that these proteins and HSA (A320T) are used as a means for stabilizing various proteins such as growth hormone to be administered to humans and other animals as pharmaceuticals in the blood after administration. It shows that it is effective to connect, in particular, it is effective to connect the C-terminal of these proteins with the N-terminal of HSA (A320T), for example, by a peptide bond.
- FIG. 7 shows the results of pharmacokinetic analysis of HSA-fused 22KhGH.
- the vertical axis represents plasma IGF-1 concentration, and the horizontal axis represents HSA-22KhGH fusion protein. It represents the elapsed time after administration.
- IGF-1 is a polypeptide having an activity such as a bone growth promoting action that is secreted by growth hormone, and it is known that a part of the physiological activity of hGH is exhibited through IGF-1.
- the plasma IGF-1 concentration is HSA-22KhGH-administered animals. Shows the maximum value about 1.5 times before the administration on the third day after administration, and shows the maximum value about twice and before the administration on the second day after administration in the HSA-m22KhGH-administered animals. However, in any case, the plasma IGF-1 concentration thereafter decreased, and after the fifth day after administration, the value was almost the same as that of the control 22KhGH. In FIG. 7, no significant increase in plasma IGF-1 concentration was observed after administration of 22KhGH.
- the blood half-life of 22KhGH was as short as about 20 minutes. This is probably because the value almost returned to that before administration.
- the plasma IGF-1 concentration in 22KhGH-administered animals increased from the second day, and was higher than that before administration until the ninth day, the last day of measurement. This is probably because the effect of 22KhGH was accumulated by continuous administration of 22KhGH only for 7 days.
- the concentration of IGF-1 in the plasma of animals administered with HSA-22KhGH or mHSA-22KhGH in which 22KhGH is linked to the N-terminal side of human serum albumin is 7 days after administration in animals administered with 22KhGH-HSA.
- the maximum value is about 2.0 times before administration, and the maximum value is about 2.0 times before administration even in the 22KhGH-mHSA-administered animals on the 7th day after administration.
- the concentration of IGF-1 in plasma was maintained at a high level even on the 9th day after administration, compared with 22KhGH as a control.
- the IGF-1 concentration of HSA-22KhGH tended to be higher until the third day after administration, but the IGF-I of mHSA-22KhGH was increased after the fifth day after administration.
- One concentration is consistently higher, indicating that mHSA-22KhGH can sustain high levels of IGF-1 in the blood over a longer period of time compared to HSA-22KhGH.
- the existing growth hormone preparation (Growject (registered) 22KhGH-mHSA in which the C-terminal side of growth hormone is linked to the N-terminal side of HSA (A320T) can be suitably used as a sustained growth hormone that lasts for a longer time than the trademark)) It shows that. Furthermore, these results indicate that the activity of these physiologically active proteins can be remarkably sustained in plasma by linking the physiologically active proteins to be administered to animals as pharmaceuticals etc. to the N-terminal side of HSA (A320T).
- HSA HSA
- 22KhGH-mHSA In animals treated with 22KhGH-mHSA, the plasma IGF-1 concentration was maintained at a very high level even on the 9th day after administration. Therefore, 22KhGH-mHSA was treated with growth hormone deficiency short stature, adult growth hormone deficiency, etc. It is reasonably predicted that even if the administration interval is 7 to 14 days, the drug efficacy will be fully exerted.
- Table 3 shows the dosage and administration when 22KhGH-mHSA is administered to patients with growth hormone deficiency short stature, adult growth hormone deficiency, and the like. The dose and administration interval shown in Table 3 should be increased or decreased as appropriate according to clinical symptoms and laboratory findings such as IGF-1 concentration. Also preferably, 22KhGH-mHSA is administered to the patient in the form of intramuscular or subcutaneous injection.
- the blood stability of a desired protein to be administered to an animal as a pharmaceutical can be increased, a new amount of the desired protein that can be reduced when administered as a pharmaceutical can be reduced.
- Drugs can be provided.
- Sequence number 3 Human serum albumin variant (A320T)
- SEQ ID NO: 4 Linker example SEQ ID NO: 5: Linker example SEQ ID NO: 6: Linker example SEQ ID NO: 8: Partial sequence of internal ribosome binding site derived from mutant mouse encephalomyocarditis virus, synthetic SEQ ID NO: 11: 22KhGH-mHSA, mature type SEQ ID NO: 12: 20 KhGH-mHSA, mature SEQ ID NO: 13: primer Hyg-Sfi5 ′, synthetic SEQ ID NO: 14: primer Hyg-BstX3 ′, synthetic SEQ ID NO: 15: IRES-Hygr-mPGKpA, synthetic SEQ ID NO: 16: resistance to hygromycin Amino acid sequence corresponding to the gene SEQ ID NO: 17: primer IRES5 ′, synthetic sequence number 18: primer IRES3 ′, synthetic sequence number 19: primer mPGKP5 ′, synthetic sequence number 20: primer mPGKP3 ′
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
1.配列番号3で示されるアミノ酸配列に対し,10個以下のアミノ酸残基が欠失し及び/又は10個以下のアミノ酸残基が置換されてなるアミノ酸配列であって,但し配列番号3で示されるアミノ酸配列のN末端から318番目のアスパラギン残基及び320番目のトレオニン残基がこれら2残基の間にプロリン以外の単一のアミノ酸残基(X)を介してペプチド結合により連結された状態で保存されているものであるアミノ酸配列を含んでなる,ヒト血清アルブミン変異体。
2.該アミノ酸残基(X)がチロシンである,上記1のヒト血清アルブミン変異体。
3.配列番号3で示されるアミノ酸配列からなるものである,請求項2のヒト血清アルブミン変異体。
4.上記1~3の何れかのヒト血清アルブミン変異体のアミノ酸配列に対し,配列番号3で示されるアミノ酸配列のN末端から318番目~320番目に対応する位置の配列部分以外において,10個以下のアミノ酸残基が付加されてなり,且つ配列番号2に示されるアミノ酸配列とは同一でないものである,ヒト血清アルブミン変異体。
5.上記1~3の何れかのヒト血清アルブミン変異体のアミノ酸配列に対し,10個以下のアミノ酸残基がN末端又はC末端に付加されてなり,且つ配列番号2に示されるアミノ酸配列とは同一でないものである,ヒト血清アルブミン変異体。
6.上記1~5の何れかのヒト血清アルブミン変異体のアミノ酸配列を含んでなる第1のポリペプチド鎖と,これに連結された他の蛋白質(A)のアミノ酸配列を含んでなる第2のポリペプチド鎖とを含んでなるものである,ヒト血清アルブミン変異体連結蛋白質(A)。
7.(a)該第1のポリペプチド鎖のN末端に該第2のポリペプチド鎖のC末端が,又は
(b)該第1のポリペプチド鎖のC末端に該第2のポリペプチド鎖のN末端が,
ペプチド結合を介して連結しているものである,上記6のヒト血清アルブミン変異体連結蛋白質(A)。
8.該ペプチド結合を介した連結が,リンカーとのペプチド結合を含むものである,上記7のヒト血清アルブミン変異体連結蛋白質。
9.該リンカーが,1~50個のアミノ酸残基からなるものである,上記8のヒト血清アルブミン変異体連結蛋白質(A)。
10.該リンカーが,1~6個のアミノ酸残基からなるものである,上記8のヒト血清アルブミン変異体連結蛋白質(A)。
11.該リンカーが,Gly-Ser,Gly-Gly-Ser,配列番号4,配列番号5,及び配列番号6で示されるアミノ酸配列からなる群より選択されるものである,上記8のヒト血清アルブミン変異体連結蛋白質。
12.該リンカーが,アミノ酸配列Gly-Serで示されるものである,上記8のヒト血清アルブミン変異体連結蛋白質(A)。
13.該蛋白質(A)が,生体に投与したときに生理活性を示すものである,上記6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
14.該蛋白質(A)が,α-L-イズロニダーゼ,イズロン酸-2-スルファターゼ,グルコセレブロシダーゼ,β-ガラクトシダーゼ,GM2活性化蛋白質,β-ヘキソサミニダーゼA,β-ヘキソサミニダーゼB,N-アセチルグルコサミン-1-ホスフォトランスフェラーゼ,α-マンノシダーゼ,β-マンノシダーゼ,ガラクトシルセラミダーゼ,サポシンC,アリルスルファターゼA,α-L-フコシダーゼ,アスパルチルグルコサミニダーゼ,α-N-アセチルガラクトサミニダーゼ,酸性スフィンゴミエリナーゼ,α-ガラクトシダーゼ,β-グルクロニダーゼ,ヘパラン硫酸N-スルファターゼ,α-N-アセチルグルコサミニダーゼ,アセチルCoAα-グルコサミニドN-アセチルトランスフェラーゼ,N-アセチルグルコサミン-6-硫酸スルファターゼ,酸性セラミダーゼ,アミロ-1,6-グルコシダーゼ,CLN1~10を含むリソソーム酵素,PD-1リガンド,骨形成蛋白質(BMP),インスリン,プロラクチン,モチリン,副腎皮質刺激ホルモン(ACTH),メラノサイト刺激ホルモン(MSH),甲状腺ホルモン放出ホルモン(TRH),甲状腺刺激ホルモン(TSH),黄体形成ホルモン(LH),卵胞刺激ホルモン(FSH),副甲状腺ホルモン(PTH)トロンボポエチン,幹細胞因子(SCF),レプチン,バソプレシン,オキシトシン,カルシトニン,グルカゴン,ガストリン,セクレチン,パンクレオザイミン,コレシストキニン,アンジオテンシン,アンジオスタチン,エンドスタチン,ヒト胎盤ラクトーゲン(HPL),ヒト絨毛性性腺刺激ホルモン(HCG),エンケファリン,エンドルフィン,インターフェロンα,インターフェロンβ,インターフェロンγ,インターロイキン2,サイモポイエチン,サイモスチムリン,胸腺液性因子(THF),血中胸腺因子(FTS),サイモシン,サイミックファクターX,腫瘍壊死因子(TNF),顆粒球コロニー刺激因子(G-CSF),マクロファージコロニー刺激因子(M-CSF),顆粒球マクロファージコロニー刺激因子(GM-CSF),ウロキナーゼ,組織プラスミノーゲン活性化因子(tPA),ダイノルフィン,ボンベシン,ニューロテンシン,セルレイン,ブラディキニン,アスパラギナーゼ,カリクレイン,サブスタンスP,神経成長因子(NGF),毛様体神経栄養因子(CNTF),脳由来神経栄養因子(BDNF),グリア細胞株由来神経栄養因子(GDNF),ニューロトロフィン3,ニューロトロフィン4/5,ニューロトロフィン6,ニューレグリン1,アクチビン,塩基性線維芽細胞成長因子(bFGF),線維芽細胞成長因子2(FGF2),血管内皮増殖因子(VEGF),骨形成蛋白質(BMP),巨核球増殖分化因子(MGDF),血液凝固因子VII,血液凝固因子VIII,血液凝固因子IX,スーパーオキシドジスムターゼ(SOD),塩酸リゾチーム,ポリミキシンB,コリスチン,グラミシジン,バシトラシン,胃酸分泌抑制ポリペプチド(GIP),血管作動性腸ポリペプチド(VIP),血小板由来成長因子(PDGF),成長ホルモン分泌因子(GRF),上皮細胞成長因子(EGF),エリスロポエチン,ソマトスタチン,インスリン様成長因子1(IGF-1),20K成長ホルモン,22K成長ホルモン,及びこれらの塩若しくは変異体からなる群より選択されるものである,上記6~13の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
15.該蛋白質(A)が,22K成長ホルモンである,上記6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
16.該蛋白質(A)が,20K成長ホルモンである,上記6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
17.配列番号11で示されるアミノ酸配列からなるものである,上記15のヒト血清アルブミン変異体連結蛋白質(A)。
18.配列番号12で示されるアミノ酸配列からなるものである,上記16のヒト血清アルブミン変異体連結蛋白質(A)。
19.上記6~18の何れかのヒト血清アルブミン変異体連結蛋白質(A)を有効成分として含有してなる,医薬。
20.成長ホルモン分泌不全性低身長症,ターナー症候群における低身長,慢性腎不全による低身長症,プラダーウィリー症候群における低身長症,軟骨異栄養症における低身長症,及びSGA性低身長症であって,何れも骨端線閉鎖を伴わないもの,並びに成人成長ホルモン分泌不全症,AIDSによる消耗,及び拒食症による消耗からなる群より選択される疾患の治療剤である,上記19の医薬。
21.上記1~5の何れかのヒト血清アルブミン変異体をコードする遺伝子を含んでなるDNA。
22.上記6~18の何れかのヒト血清アルブミン変異体連結蛋白質(A)をコードする遺伝子を含んでなるDNA。
23.上記21又は22のDNAを含んでなる発現ベクター。
24.上記23のベクターで形質転換された哺乳類細胞。
25.上記24の哺乳類細胞を,無血清培地で培養することにより得られる,ヒト血清アルブミン変異体又はヒト血清アルブミン変異体連結蛋白質(A)。
pEF/myc/nucベクター(インビトロジェン社)を,制限酵素(KpnI及びNcoI)で消化し,EF-1αプロモーター及びその第一イントロンを含むDNA断片を切り出し,このDNA断片をT4 DNAポリメラーゼで平滑末端化処理した。別に,pCI-neo(インビトロジェン社)を,制限酵素(BglII及びEcoRI)で消化して,CMVのエンハンサー/プロモーター及びイントロンを含む領域を切除した後に,T4 DNAポリメラーゼで平滑末端化処理した。これに,上記のEF-1αプロモーター及びその第一イントロンを含む領域(平滑末端化処理後のもの)を挿入して,pE-neoベクターを構築した(図1)。
野生型HSA(配列番号1)のC末端と22KhGHのN末端とを融合させたものである融合蛋白質HSA-22KhGHのアミノ酸配列を,配列番号32に示す。このアミノ酸配列中,1~585番目のアミノ酸残基が野生型の成熟HSAのアミノ酸配列(配列番号1)に相当し,586~776番目のアミノ酸残基が22KhGHのアミノ酸配列に相当する。HSA-22KhGHをコードする遺伝子(HSA-22KhGH遺伝子)を含む配列番号33で示した塩基配列を有するDNAを化学的に合成した。この配列において,塩基11~82がHSAのリーダーペプチドを,塩基83~1837が成熟型HSAを,そして塩基1838~2410が成熟型hGHを,それぞれコードする。このDNAを制限酵素(MluI及びNotI)で消化し,実施例1で作製したpE-mIRES-GS-puroのMluI部位とNotI部位の間に組み込むことにより,HSA-22KhGH発現用ベクターpE-mIRES-GS-puro(HSA-22KhGH)を構築した。
HSA(A320T)(配列番号3)のC末端と22KhGHのN末端とを融合させたものである,配列番号34で示すアミノ酸配列を有する融合蛋白質を,mHSA-22KhGHとした。配列番号34で示すアミノ酸配列中,1~585番目のアミノ酸残基がmHSAのアミノ酸配列に相当し,586~776番目のアミノ酸残基が22KhGHのアミノ酸配列に相当する。実施例2で作製したpE-mIRES-GS-puro(HSA-22KhGH)を鋳型として,プライマーYA082(配列番号35)及びプライマーYA083(配列番号36)を用いて,PCRによりmHSA-22KhGHをコードする遺伝子を含むDNA断片を増幅した。このDNA断片をセルフアニールさせて,mHSA-22KhGH発現用ベクターであるpE-mIRES-GS-puro(mHSA-22KhGH)を構築した。
22KhGHのC末端と野生型HSA(配列番号1)のN末端とを融合させたものである,配列番号37で示したアミノ酸配列を有する融合蛋白質を,22KhGH-HSAとした。配列番号37で示したアミノ酸配列中,1~191番目のアミノ酸残基が22KhGHのアミノ酸配列に相当し,192~776番目のアミノ酸残基がHSAのアミノ酸配列に相当する。22KhGH-HSAをコードする遺伝子(22KhGH-HSA遺伝子)を含む配列番号38で示した塩基配列を有するDNAを化学的に合成した。この配列において,塩基11~88がhGHのリーダーペプチドを,塩基89~661が成熟型hGHを,塩基662~2416が成熟型HSAを,それぞれコードする。このDNAを制限酵素(MluI及びNotI)で消化し,実施例1で作製したpE-mIRES-GS-puroのMluI部位とNotI部位の間に組み込むことにより,HSA-22KhGH発現用ベクターであるpE-mIRES-GS-puro(22KhGH-HSA)を構築した。
22KhGHのC末端とHSA(A320T)(配列番号3)のN末端とを融合させたものである,配列番号39に示したアミノ酸配列を有する融合蛋白質を,22KhGH-mHSAとした。実施例4で作製したpE-mIRES-GS-puro(22KhGH-HSA)を鋳型として,プライマーYA082(配列番号35)及びプライマーYA083(配列番号36)を用いて,PCRにより22KhGH-mHSAをコードする遺伝子を含むDNA断片を増幅した。このDNA断片をセルフアニールさせて,22KhGH-mHSA発現用ベクターであるpE-mIRES-GS-puro(22KhGH-mHSA)を構築した。
HSA-22KhGH,mHSA-22KhGH,22KhGH-HSA,及び22KhGH-mHSAの各融合蛋白質を発現させるための細胞を次のようにして作製した。チャイニーズハムスターの卵巣に由来する細胞であるCHO-K1細胞に,Gene Pulser Xcell エレクトロポレーションシステム(Bio Rad社)を用いて,実施例2~5で作製した,HSA-22KhGH発現用ベクターpE-mIRES-GS-puro(HSA-22KhGH),mHSA-22KhGH発現用ベクターpE-mIRES-GS-puro(mHSA-22KhGH),22KhGH-HSA発現用ベクターpE-mIRES-GS-puro(22KhGH-HSA),及び22KhGH-mHSA発現用ベクターpE-mIRES-GS-puro(22KhGH-mHSA)をそれぞれ導入した。それぞれの発現ベクターを導入した細胞を,メチオニンスルホキシミン(SIGMA社)及びピューロマイシン(SIGMA社)を含むCD OptiCHOTM培地(Thermo Fisher Scientific社)を用いて選択培養し,HSA-22KhGH発現細胞,mHSA-22KhGH発現細胞,22KhGH-HSA発現細胞,及び22KhGH-mHSA発現細胞をそれぞれ確立した。選択培養の際には,メチオニンスルホキシミン及びピューロマイシンの濃度を段階的に上昇させて,最終的にメチオニンスルホキシミンの濃度を300μM,ピューロマイシンの濃度を10μg/mLとして,より高い薬剤耐性を示す細胞を選択的に増殖させた。
HSA-22KhGH発現細胞,mHSA-22KhGH発現細胞,22KhGH-HSA発現細胞,及び22KhGH-mHSA発現細胞の培養を,次のようにして行った。CD OptiCHOTM培地(Thermo Fisher Scientific社)に,メチオニンスルホキシミンとピューロマイシンとを,それぞれ300μM及び10μg/mLの濃度となるように添加して,細胞培養用培地を調製した。実施例6で作製した各発現細胞を,それぞれ2×105 個/mLの細胞密度となるように5mLの細胞培養用培地に添加し,5%CO2存在下37℃で培養した。5日毎に,2×105 個/mLの細胞密度となるように,細胞を新しい培養用培地に移して,継代培養を行った。
HSA-22KhGH,mHSA-22KhGH,22KhGH-HSA,及び22KhGH-mHSAの精製を次のようにして行った。実施例7で継代培養した各発現細胞を,それぞれ細胞培養用培地に全量が240mLとなるように2×105個/mLの密度で懸濁した。この細胞懸濁液を30mLずつ,径15cmのシャーレ8枚に加えて,5日間,5%CO2存在下,37℃で培養した。培養終了後に培地を回収し,膜フィルター(孔径0.22μm,Millipore社)でろ過して,培養上清とした。次いで,各培養上清に,1M HEPES(pH8.0)を加えて,pHを7.0~7.2に調整した。
ヒトGH受容体(hGHR)遺伝子をマウスBaF3細胞に導入することによりGH依存性増殖能を獲得させたBaF3/hGHR細胞を,以下の通りにして作製した。配列番号40に示した塩基配列を有するhGHR ECD人工合成遺伝子(hGHRの細胞外領域(Extra Cellular Domain)をコードするhGHR遺伝子の5’側断片)を鋳型として,プライマーYA034(配列番号41)及びプライマーYA035(配列番号42)を用いてPCRを行った。得られたPCR産物をアガロース電気泳動に供し,QIAEX II(QIAGEN社)を用いて精製した。このDNA断片をメガプライマーとした。ヒト肺由来cDNAを鋳型として,プライマーK708(配列番号43)及びプライマーK709(配列番号44)を用いてPCRを行い,hGHR遺伝子の全長を含むDNA断片を増幅した。得られたPCR産物をアガロース電気泳動に供し,QIAEX IIを用いて精製した。次いで,精製したhGHR遺伝子の全長を含むDNA断片を鋳型として,上記メガプライマー及びプライマーK709(配列番号44)を用いてPCRを行い,5’側にhGHR ECD人工合成遺伝子配列を有するhGHRの全長をコードする遺伝子を含む,配列番号45に示した塩基配列を有するDNA断片を増幅した。このDNA断片を制限酵素(MluI及びNotI)で消化し,レトロウイルスベクターであるpMX-II(Ono Y., Oncogene. 19. 3050-8(2000))のMluIとNotIの間に組み込み,これをhGHR発現用レトロウイルスベクター(hGHR/pMX-II)とした。
HSA-hGH融合蛋白質の細胞増殖活性を,実施例9に記載の方法で作製したBaF3/hGHR細胞を用いて評価した。
実施例8で精製したHSA-hGH融合蛋白質(HSA-22KhGH,mHSA-22KhGH,22KhGH-HSA及び22KhGH-mHSA)を,それぞれ,4.0mg/kgの用量で雄性カニクイザルに単回皮下投与した。なお,HSA-22KhGHの投与は3匹のカニクイザルを用いて行い,mHSA-22KhGH,22KhGH-HSA及び22KhGH-mHSAの投与については,それぞれ1匹のカニクイザルを用いて行った。
マウス抗HSAモノクローナル抗体及びマウス抗hGH抗体を,当業者に周知の手法により,HSA又はhGHで免疫したマウスの脾細胞をそれぞれミエローマ細胞と融合させて得たハイブリドーマ細胞を培養して得た。マウス抗hGHモノクローナル抗体を,0.1M NaHCO3溶液(pH9)で透析した後,溶液中の抗体濃度をNanoDropTM(Thermo Scientific社)を用いて測定した。次いで,5mg/mLの濃度でDMSOに溶解したEZ-LinkTM NHS-LC-Biotin(Thermo Fisher Scientific社)を,1mgの抗体当たり60μgのNHS-LC-Biotinの比率で抗体溶液に添加し,室温で2時間反応させた後,反応溶液をPBSで透析しビオチン化マウス抗hGHモノクローナル抗体を得た。下記の定量法において,マウス抗HSAモノクローナル抗体を一次抗体,ビオチン化マウス抗hGHモノクローナル抗体を二次抗体として使用した。
測定は,Sector Imager 6000の使用説明書に従って,概ね下記のとおり実施した。マウス抗HSAモノクローナル抗体をHigh Bind Plate(Meso Scale Diagnostics社)に添加し,1時間静置して抗HSA抗体(一次抗体)をプレートに固定した。次いで,Superblock Blocking buffer in PBS(Thermo Fisher Scientific社)をプレートに添加して1時間振盪し,プレートをブロッキングした。プレートをPBST(0.05% Tween20を含有するPBS)で洗浄した後,検体を添加して1時間振盪した。プレートをPBSTで洗浄した後,ビオチン化マウス抗hGHモノクローナル抗体(二次抗体)を添加して1時間振盪した。プレートをPBSTで洗浄した後,SULFO-Tag-Streptavidin(Meso Scale Diagnostics社)を添加して1時間振盪した。プレートをPBSTで洗浄した後,Read buffer T(Meso Scale Diagnostics社)を添加し,Sector Imager 6000(Meso Scale Diagnostics社)で波長620nmの発光を測定した。同様にして同一プレート上で既知濃度のHSA-hGHを定量して標準曲線を求め,これに検体の測定値を内挿し,血漿中のHSA-hGH融合蛋白質の濃度を求めた。
血漿中に含まれるIGF-1の定量は,Human IGF-I Quantikine ELISA kit(R&D systems)を用いて,ELISA法により行った。
(1)BaF3/hGHR細胞を用いた細胞増殖活性の測定
図5は,縦軸に490nmにおける吸光度,横軸に各検体のモル濃度(nM)をとり測定値をプロットした,BaF3/hGHR細胞を用いた細胞増殖活性の測定結果を示す図である。この図から,各検体のEC50を求めた結果を表1に示す。
図6は,縦軸にカニクイザル血清中のHSA-hGH融合蛋白質(HSA-22KhGH,mHSA-22KhGH,22KhGH-HSA及び22KhGH-mHSA)の濃度,横軸にHSA-hGH融合蛋白質を投与後の経過時間をプロットした,HSA-hGH融合蛋白質の薬物動態解析の結果を示す図である。この図から,各検体のCmax,AUC0-216h,AUC0-inf及びt1/2βを求めた結果を表2に示す。
図7は,HSA融合22KhGHの薬物動態解析の結果を示す図であり,縦軸は血漿中のIGF-1の濃度を,横軸はHSA-22KhGH融合蛋白質を投与後の経過時間を表す。IGF-1は成長ホルモンによって分泌が誘導される骨成長促進作用等の活性を有するポリペプチドであり,hGHの生理活性の一部はIGF-1を介して発揮されることが知られている。
リン酸水素二ナトリウム七水和物・・・・1.33mg
リン酸二水素ナトリウム・・・・・・・・1.57mg
ポリオキシエチレン(160)ポリオキシ
プロピレン(30)グリコール・・・・・3mg
ベンジルアルコール・・・・・・・・・13.5mg
D-マンニトール・・・・・・・・・・52.5mg
22KhGH-mHSA・・・・・・・・1mg
上記成分比率で各成分を注射用水に溶解させ,pHを6.0~6.4に調整し,液量を1.5mLとして水性注射剤とする。
L-ヒスチジン・・・・・・・・・・・・1mg
フェノール・・・・・・・・・・・・・・4.5mg
ポリオキシエチレン(160)ポリオキシ
プロピレン(30)グリコール・・・・・4.5mg
D-マンニトール・・・・・・・・・・60mg
22KhGH-mHSA・・・・・・・・1mg
上記成分比率で各成分を注射用水に溶解させて,pHを6.0~6.4に調整し,液量を1.5mLとして水性注射剤とする。
リン酸水素二ナトリウム七水和物・・・・2.475mg
リン酸二水素ナトリウム・・・・・・・・0.394mg
塩化ナトリウム・・・・・・・・・・・1.125mg
アミノ酢酸・・・・・・・・・・・・・11.25mg
D-マンニトール・・・・・・・・・・22.5mg
22KhGH-mHSA・・・・・・・・1mg
上記成分からなる凍結乾燥剤を,使用時に9.7mgのベンジルアルコールを含有する1mLの注射用水に溶解する。
2 mPGKプロモーター
3 配列番号7に示す塩基配列を含む野生型マウス脳心筋炎ウイルスの内部リボソーム結合部位の部分配列
3a 配列番号8に示す塩基配列を含む変異型マウス脳心筋炎ウイルスの内部リボソーム結合部位の部分配列
4 mPGKのポリアデニル化領域(mPGKpA)
5 EF-1p及び第一イントロンを含む塩基配列
6 SV40後期ポリアデニル化領域
7 SV40初期プロモーターを含む領域
8 合成ポリアデニル化領域
9 サイトメガロウイルスプロモーターを含む領域
10 グルタミン合成酵素遺伝子
配列番号4:リンカー例
配列番号5:リンカー例
配列番号6:リンカー例
配列番号8:変異型マウス脳心筋炎ウイルス由来の内部リボソーム結合部位の部分配列,合成
配列番号11:22KhGH-mHSA,成熟型
配列番号12:20KhGH-mHSA,成熟型
配列番号13:プライマーHyg-Sfi5',合成
配列番号14:プライマーHyg-BstX3',合成
配列番号15:IRES-Hygr-mPGKpA,合成
配列番号16:ハイグロマイシン耐性遺伝子に対応するアミノ酸配列
配列番号17:プライマーIRES5',合成
配列番号18:プライマーIRES3',合成
配列番号19:プライマーmPGKP5',合成
配列番号20:プライマーmPGKP3',合成
配列番号21:mPGKp,合成
配列番号22:プライマーGS5',合成
配列番号23:プライマーGS3',合成
配列番号24:プライマーpuro5',合成
配列番号25:プライマーpuro3',合成
配列番号26:ピューロマイシン耐性遺伝子を含む配列
配列番号27:ピューロマイシン耐性遺伝子に対応するアミノ酸配列
配列番号28:プライマーSV40polyA5',合成
配列番号29:プライマーSV40polyA3',合成
配列番号30:プライマーmIRES-GS5',合成
配列番号31:プライマーmIRES-GS3',合成
配列番号32:HSA-22KhGH,成熟型
配列番号33:HSA-22KhGH遺伝子を含む配列,合成
配列番号34:mHSA-22KhGH,成熟型
配列番号35:プライマーYA082,合成配列
配列番号36:プライマーYA083,合成配列
配列番号37:22KhGH-HSA,成熟型
配列番号38:22KhGH-HSA遺伝子を含む配列,合成
配列番号39:22KhGH-mHSA
配列番号40:hGHR ECDをコードする人工合成遺伝子の配列
配列番号41:プライマーYA034,合成
配列番号42:プライマーYA035,合成
配列番号43:プライマーK708,合成
配列番号44:プライマーK709,合成
配列番号45:hGHRをコードする人工合成遺伝子の塩基配列,合成
Claims (25)
- 配列番号3で示されるアミノ酸配列に対し,10個以下のアミノ酸残基が欠失し及び/又は10個以下のアミノ酸残基が置換されてなるアミノ酸配列であって,但し配列番号3で示されるアミノ酸配列のN末端から318番目のアスパラギン残基及び320番目のトレオニン残基がこれら2残基の間にプロリン以外の単一のアミノ酸残基(X)を介してペプチド結合により連結された状態で保存されているものであるアミノ酸配列を含んでなる,ヒト血清アルブミン変異体。
- 該アミノ酸残基(X)がチロシンである,請求項1のヒト血清アルブミン変異体。
- 配列番号3で示されるアミノ酸配列からなるものである,請求項2のヒト血清アルブミン変異体。
- 請求項1~3の何れかのヒト血清アルブミン変異体のアミノ酸配列に対し,配列番号3で示されるアミノ酸配列のN末端から318番目~320番目に対応する位置の配列部分以外において,10個以下のアミノ酸残基が付加されてなり,且つ配列番号2に示されるアミノ酸配列とは同一でないものである,ヒト血清アルブミン変異体。
- 請求項1~3の何れかのヒト血清アルブミン変異体のアミノ酸配列に対し,10個以下のアミノ酸残基がN末端又はC末端に付加されてなり,且つ配列番号2に示されるアミノ酸配列とは同一でないものである,ヒト血清アルブミン変異体。
- 請求項1~5の何れかのヒト血清アルブミン変異体のアミノ酸配列を含んでなる第1のポリペプチド鎖と,これに連結された他の蛋白質(A)のアミノ酸配列を含んでなる第2のポリペプチド鎖とを含んでなるものである,ヒト血清アルブミン変異体連結蛋白質(A)。
- (a)該第1のポリペプチド鎖のN末端に該第2のポリペプチド鎖のC末端が,又は
(b)該第1のポリペプチド鎖のC末端に該第2のポリペプチド鎖のN末端が,
ペプチド結合を介して連結しているものである,請求項6のヒト血清アルブミン変異体連結蛋白質(A)。 - 該ペプチド結合を介した連結が,リンカーとのペプチド結合を含むものである,請求項7のヒト血清アルブミン変異体連結蛋白質。
- 該リンカーが,1~50個のアミノ酸残基からなるものである,請求項8のヒト血清アルブミン変異体連結蛋白質(A)。
- 該リンカーが,1~6個のアミノ酸残基からなるものである,請求項8のヒト血清アルブミン変異体連結蛋白質(A)。
- 該リンカーが,Gly-Ser,Gly-Gly-Ser,配列番号4,配列番号5,及び配列番号6で示されるアミノ酸配列からなる群より選択されるものである,請求項8のヒト血清アルブミン変異体連結蛋白質。
- 該リンカーが,アミノ酸配列Gly-Serで示されるものである,請求項8のヒト血清アルブミン変異体連結蛋白質(A)。
- 該蛋白質(A)が,生体に投与したときに生理活性を示すものである,請求項6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
- 該蛋白質(A)が,α-L-イズロニダーゼ,イズロン酸-2-スルファターゼ,グルコセレブロシダーゼ,β-ガラクトシダーゼ,GM2活性化蛋白質,β-ヘキソサミニダーゼA,β-ヘキソサミニダーゼB,N-アセチルグルコサミン-1-ホスフォトランスフェラーゼ,α-マンノシダーゼ,β-マンノシダーゼ,ガラクトシルセラミダーゼ,サポシンC,アリルスルファターゼA,α-L-フコシダーゼ,アスパルチルグルコサミニダーゼ,α-N-アセチルガラクトサミニダーゼ,酸性スフィンゴミエリナーゼ,α-ガラクトシダーゼ,β-グルクロニダーゼ,ヘパラン硫酸N-スルファターゼ,α-N-アセチルグルコサミニダーゼ,アセチルCoAα-グルコサミニドN-アセチルトランスフェラーゼ,N-アセチルグルコサミン-6-硫酸スルファターゼ,酸性セラミダーゼ,アミロ-1,6-グルコシダーゼ,CLN1~10を含むリソソーム酵素,PD-1リガンド,骨形成蛋白質(BMP),インスリン,プロラクチン,モチリン,副腎皮質刺激ホルモン(ACTH),メラノサイト刺激ホルモン(MSH),甲状腺ホルモン放出ホルモン(TRH),甲状腺刺激ホルモン(TSH),黄体形成ホルモン(LH),卵胞刺激ホルモン(FSH),副甲状腺ホルモン(PTH)トロンボポエチン,幹細胞因子(SCF),レプチン,バソプレシン,オキシトシン,カルシトニン,グルカゴン,ガストリン,セクレチン,パンクレオザイミン,コレシストキニン,アンジオテンシン,アンジオスタチン,エンドスタチン,ヒト胎盤ラクトーゲン(HPL),ヒト絨毛性性腺刺激ホルモン(HCG),エンケファリン,エンドルフィン,インターフェロンα,インターフェロンβ,インターフェロンγ,インターロイキン2,サイモポイエチン,サイモスチムリン,胸腺液性因子(THF),血中胸腺因子(FTS),サイモシン,サイミックファクターX,腫瘍壊死因子(TNF),顆粒球コロニー刺激因子(G-CSF),マクロファージコロニー刺激因子(M-CSF),顆粒球マクロファージコロニー刺激因子(GM-CSF),ウロキナーゼ,組織プラスミノーゲン活性化因子(tPA),ダイノルフィン,ボンベシン,ニューロテンシン,セルレイン,ブラディキニン,アスパラギナーゼ,カリクレイン,サブスタンスP,神経成長因子(NGF),毛様体神経栄養因子(CNTF),脳由来神経栄養因子(BDNF),グリア細胞株由来神経栄養因子(GDNF),ニューロトロフィン3,ニューロトロフィン4/5,ニューロトロフィン6,ニューレグリン1,アクチビン,塩基性線維芽細胞成長因子(bFGF),線維芽細胞成長因子2(FGF2),血管内皮増殖因子(VEGF),骨形成蛋白質(BMP),巨核球増殖分化因子(MGDF),血液凝固因子VII,血液凝固因子VIII,血液凝固因子IX,スーパーオキシドジスムターゼ(SOD),塩酸リゾチーム,ポリミキシンB,コリスチン,グラミシジン,バシトラシン,胃酸分泌抑制ポリペプチド(GIP),血管作動性腸ポリペプチド(VIP),血小板由来成長因子(PDGF),成長ホルモン分泌因子(GRF),上皮細胞成長因子(EGF),エリスロポエチン,ソマトスタチン,インスリン様成長因子1(IGF-1),20K成長ホルモン,22K成長ホルモン,及びこれらの塩若しくは変異体からなる群より選択されるものである,請求項6~13の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
- 該蛋白質(A)が,22K成長ホルモンである,請求項6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
- 該蛋白質(A)が,20K成長ホルモンである,請求項6~12の何れかのヒト血清アルブミン変異体連結蛋白質(A)。
- 配列番号11で示されるアミノ酸配列からなるものである,請求項15のヒト血清アルブミン変異体連結蛋白質(A)。
- 配列番号12で示されるアミノ酸配列からなるものである,請求項16のヒト血清アルブミン変異体連結蛋白質(A)。
- 請求項6~18の何れかのヒト血清アルブミン変異体連結蛋白質(A)を有効成分として含有してなる,医薬。
- 成長ホルモン分泌不全性低身長症,ターナー症候群における低身長,慢性腎不全による低身長症,プラダーウィリー症候群における低身長症,軟骨異栄養症における低身長症,及びSGA性低身長症であって,何れも骨端線閉鎖を伴わないもの,並びに成人成長ホルモン分泌不全症,AIDSによる消耗,及び拒食症による消耗からなる群より選択される疾患の治療剤である,請求項19の医薬。
- 請求項1~5の何れかのヒト血清アルブミン変異体をコードする遺伝子を含んでなるDNA。
- 請求項6~18の何れかのヒト血清アルブミン変異体連結蛋白質(A)をコードする遺伝子を含んでなるDNA。
- 請求項21又は22のDNAを含んでなる発現ベクター。
- 請求項23のベクターで形質転換された哺乳類細胞。
- 請求項24の哺乳類細胞を,無血清培地で培養することにより得られる,ヒト血清アルブミン変異体又はヒト血清アルブミン変異体連結蛋白質ヒト血清アルブミン変異体連結蛋白質(A)。
Priority Applications (21)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES16844430T ES2861059T3 (es) | 2015-09-08 | 2016-09-08 | Nuevo mutante de la seroalbúmina humana |
DK16844430.5T DK3348635T3 (da) | 2015-09-08 | 2016-09-08 | Hidtil ukendt mutant af humant serumalbumin |
PL16844430T PL3348635T3 (pl) | 2015-09-08 | 2016-09-08 | Nowy mutant albuminy krwi ludzkiej |
KR1020187009487A KR102360744B1 (ko) | 2015-09-08 | 2016-09-08 | 신규 인간 혈청 알부민 변이체 |
EP16844430.5A EP3348635B1 (en) | 2015-09-08 | 2016-09-08 | Novel human serum albumin mutant |
JP2017539213A JP6886920B2 (ja) | 2015-09-08 | 2016-09-08 | 新規なヒト血清アルブミン変異体 |
US15/758,199 US10654912B2 (en) | 2015-09-08 | 2016-09-08 | Human serum albumin mutant |
CA2996672A CA2996672C (en) | 2015-09-08 | 2016-09-08 | Novel human serum albumin mutant |
SI201631160T SI3348635T1 (sl) | 2015-09-08 | 2016-09-08 | Novi mutant humanega serumskega albumina |
CN202210350644.8A CN114591445B (zh) | 2015-09-08 | 2016-09-08 | 新型人血清白蛋白突变体 |
EP24163473.2A EP4374913A2 (en) | 2015-09-08 | 2016-09-08 | Novel human serum albumin mutant |
EP20216964.5A EP3845644B1 (en) | 2015-09-08 | 2016-09-08 | Novel human serum albumin mutant |
CN201680051790.3A CN107949638B (zh) | 2015-09-08 | 2016-09-08 | 新型人血清白蛋白突变体 |
AU2016319540A AU2016319540B2 (en) | 2015-09-08 | 2016-09-08 | Novel human serum albumin mutant |
HK18106822.1A HK1247244A1 (zh) | 2015-09-08 | 2018-05-25 | 新型人血清白蛋白突變體 |
US16/846,003 US11046751B2 (en) | 2015-09-08 | 2020-04-10 | Human serum albumin mutant |
CY20211100290T CY1123997T1 (el) | 2015-09-08 | 2021-04-05 | Νεα μεταλλακτικη παραλλαγη αλβουμινης ανθρωπινου ορου |
HRP20210635TT HRP20210635T1 (hr) | 2015-09-08 | 2021-04-22 | Novi mutant humanog seruma albumina |
US17/323,733 US11634474B2 (en) | 2015-09-08 | 2021-05-18 | Human serum albumin mutant |
AU2021212110A AU2021212110B2 (en) | 2015-09-08 | 2021-08-06 | Novel human serum albumin mutant |
AU2022204463A AU2022204463B2 (en) | 2015-09-08 | 2022-06-24 | Novel human serum albumin mutant |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015177093 | 2015-09-08 | ||
JP2015-177093 | 2015-09-08 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/758,199 A-371-Of-International US10654912B2 (en) | 2015-09-08 | 2016-09-08 | Human serum albumin mutant |
US16/846,003 Division US11046751B2 (en) | 2015-09-08 | 2020-04-10 | Human serum albumin mutant |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2017043569A1 true WO2017043569A1 (ja) | 2017-03-16 |
Family
ID=58239798
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2016/076438 WO2017043569A1 (ja) | 2015-09-08 | 2016-09-08 | 新規なヒト血清アルブミン変異体 |
Country Status (17)
Country | Link |
---|---|
US (3) | US10654912B2 (ja) |
EP (3) | EP3348635B1 (ja) |
JP (4) | JP6886920B2 (ja) |
KR (1) | KR102360744B1 (ja) |
CN (2) | CN114591445B (ja) |
AU (3) | AU2016319540B2 (ja) |
CA (2) | CA3183289A1 (ja) |
CY (1) | CY1123997T1 (ja) |
DK (1) | DK3348635T3 (ja) |
ES (1) | ES2861059T3 (ja) |
HK (1) | HK1247244A1 (ja) |
HR (1) | HRP20210635T1 (ja) |
HU (1) | HUE053937T2 (ja) |
PL (1) | PL3348635T3 (ja) |
PT (1) | PT3348635T (ja) |
SI (1) | SI3348635T1 (ja) |
WO (1) | WO2017043569A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017159540A1 (ja) * | 2016-03-14 | 2017-09-21 | Jcrファーマ株式会社 | 血清アルブミン-20k成長ホルモン融合タンパク質 |
WO2021075526A1 (ja) * | 2019-10-17 | 2021-04-22 | Jcrファーマ株式会社 | 血清アルブミンと成長ホルモンの融合蛋白質の製造方法 |
EP3811962A4 (en) * | 2018-06-25 | 2022-03-16 | JCR Pharmaceuticals Co., Ltd. | PROTEIN AQUEOUS LIQUID FORMULATION |
WO2024080305A1 (ja) * | 2022-10-11 | 2024-04-18 | Jcrファーマ株式会社 | 血清アルブミンと生理活性を有する蛋白質との融合蛋白質 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109734816A (zh) * | 2019-03-12 | 2019-05-10 | 王大勇 | 一种基因重组人促皮质素与白蛋白的融合蛋白及表达方法 |
WO2021085518A1 (ja) * | 2019-10-30 | 2021-05-06 | Jcrファーマ株式会社 | 血清アルブミンと成長ホルモンの融合蛋白質を含有する水性医薬組成物 |
CN113430227A (zh) * | 2020-03-23 | 2021-09-24 | 佛山汉腾生物科技有限公司 | 制备稳定细胞池的方法、蛋白表达方法及试剂盒 |
CN114133458B (zh) * | 2021-12-08 | 2023-11-14 | 福州大学 | 一种在人血清白蛋白内部融合多肽的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000502901A (ja) * | 1995-12-30 | 2000-03-14 | デルタ、バイオテクノロジー、リミテッド | 成長ホルモンおよび血清アルブミンに対する組換え融合タンパク質 |
JP2005514060A (ja) * | 2001-12-21 | 2005-05-19 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合タンパク質 |
JP2007522806A (ja) * | 2004-02-09 | 2007-08-16 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合蛋白質 |
JP2008043285A (ja) * | 2006-08-18 | 2008-02-28 | Nipro Corp | 糖鎖含有アルブミン、その製造方法およびその用途 |
Family Cites Families (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2650598B1 (fr) | 1989-08-03 | 1994-06-03 | Rhone Poulenc Sante | Derives de l'albumine a fonction therapeutique |
FR2686900B1 (fr) | 1992-01-31 | 1995-07-21 | Rhone Poulenc Rorer Sa | Nouveaux polypeptides ayant une activite de stimulation des colonies de granulocytes, leur preparation et compositions pharmaceutiques les contenant. |
FR2686899B1 (fr) | 1992-01-31 | 1995-09-01 | Rhone Poulenc Rorer Sa | Nouveaux polypeptides biologiquement actifs, leur preparation et compositions pharmaceutiques les contenant. |
JP3398427B2 (ja) * | 1992-09-09 | 2003-04-21 | 三井化学株式会社 | 成長ホルモンの生産方法 |
NZ286884A (en) * | 1995-06-29 | 1997-12-19 | Mitsui Chemicals Inc | Use of 20 kd human growth hormone in hrt, increasing serum igf-1 levels and stimulating lipolysis |
WO1997024450A1 (en) * | 1996-01-02 | 1997-07-10 | Paik Kye Hyung | Method for immunizing and treating human cells in animal organs maintained in vitro |
US6123775A (en) | 1999-06-30 | 2000-09-26 | Lam Research Corporation | Reaction chamber component having improved temperature uniformity |
AU2001259063A1 (en) | 2000-04-12 | 2001-10-30 | Human Genome Sciences, Inc. | Albumin fusion proteins |
EP1471928A1 (en) | 2002-02-07 | 2004-11-03 | Delta Biotechnology Limited | Hiv inhibiting proteins |
GB0217347D0 (en) * | 2002-07-26 | 2002-09-04 | Univ Edinburgh | Novel albumins |
CN1980687B (zh) * | 2004-02-09 | 2015-05-13 | 人类基因科学公司 | 清蛋白融合蛋白 |
US7998930B2 (en) | 2004-11-04 | 2011-08-16 | Hanall Biopharma Co., Ltd. | Modified growth hormones |
CN1313494C (zh) * | 2005-08-29 | 2007-05-02 | 中国人民解放军军事医学科学院生物工程研究所 | 一种具有促增长作用的融合蛋白及其编码基因与应用 |
JP5256199B2 (ja) | 2006-08-07 | 2013-08-07 | テヴァ バイオファーマシューティカルズ ユーエスエー,インコーポレーティッド | アルブミン−インスリン融合タンパク質 |
US7773341B2 (en) | 2007-07-03 | 2010-08-10 | Headway Technologies, Inc. | Laminated film for head applications |
US7943570B2 (en) * | 2007-10-16 | 2011-05-17 | Nipro Corporation | Sugar chain-containing albumin, production method thereof and use thereof |
CN101429240A (zh) * | 2007-11-06 | 2009-05-13 | 尼普洛株式会社 | 一种含有糖链的白蛋白、其制备方法及其用途 |
CA2611540C (en) * | 2007-11-09 | 2017-05-30 | Nipro Corporation | Sugar chain-containing albumin as a drug carrier to the liver |
BRPI1006443B1 (pt) * | 2009-04-22 | 2021-05-25 | Alteogen, Inc | Proteína ou peptídeo de fusão e método para aumentar a meia-vida in vivo de uma proteína ou peptídeo |
US8841249B2 (en) | 2009-08-06 | 2014-09-23 | Novo Nordisk A/S | Growth hormones with prolonged in-vivo efficacy |
TWI508737B (zh) | 2010-01-22 | 2015-11-21 | 諾佛 儂迪克股份有限公司 | 具有延長的活體內功效的生長激素 |
CN102336828B (zh) * | 2010-07-26 | 2013-06-26 | 中国医学科学院基础医学研究所 | 一种多发性骨髓瘤特异性蛋白及其专用检测试剂盒 |
US20130244231A1 (en) | 2010-11-08 | 2013-09-19 | Jcr Pharmaceuticals Co., Ltd. | Novel expression vector |
JP6279466B2 (ja) | 2012-04-27 | 2018-02-14 | Jcrファーマ株式会社 | 新規な発現ベクター |
-
2016
- 2016-09-08 PT PT168444305T patent/PT3348635T/pt unknown
- 2016-09-08 CA CA3183289A patent/CA3183289A1/en active Pending
- 2016-09-08 CN CN202210350644.8A patent/CN114591445B/zh active Active
- 2016-09-08 DK DK16844430.5T patent/DK3348635T3/da active
- 2016-09-08 JP JP2017539213A patent/JP6886920B2/ja active Active
- 2016-09-08 WO PCT/JP2016/076438 patent/WO2017043569A1/ja active Application Filing
- 2016-09-08 HU HUE16844430A patent/HUE053937T2/hu unknown
- 2016-09-08 EP EP16844430.5A patent/EP3348635B1/en active Active
- 2016-09-08 KR KR1020187009487A patent/KR102360744B1/ko active IP Right Grant
- 2016-09-08 AU AU2016319540A patent/AU2016319540B2/en active Active
- 2016-09-08 ES ES16844430T patent/ES2861059T3/es active Active
- 2016-09-08 US US15/758,199 patent/US10654912B2/en active Active
- 2016-09-08 PL PL16844430T patent/PL3348635T3/pl unknown
- 2016-09-08 CA CA2996672A patent/CA2996672C/en active Active
- 2016-09-08 EP EP20216964.5A patent/EP3845644B1/en active Active
- 2016-09-08 SI SI201631160T patent/SI3348635T1/sl unknown
- 2016-09-08 EP EP24163473.2A patent/EP4374913A2/en active Pending
- 2016-09-08 CN CN201680051790.3A patent/CN107949638B/zh active Active
-
2018
- 2018-05-25 HK HK18106822.1A patent/HK1247244A1/zh unknown
-
2020
- 2020-04-10 US US16/846,003 patent/US11046751B2/en active Active
-
2021
- 2021-04-05 CY CY20211100290T patent/CY1123997T1/el unknown
- 2021-04-22 HR HRP20210635TT patent/HRP20210635T1/hr unknown
- 2021-05-16 JP JP2021082852A patent/JP7123216B2/ja active Active
- 2021-05-18 US US17/323,733 patent/US11634474B2/en active Active
- 2021-08-06 AU AU2021212110A patent/AU2021212110B2/en active Active
-
2022
- 2022-06-24 AU AU2022204463A patent/AU2022204463B2/en active Active
- 2022-08-04 JP JP2022125052A patent/JP7403595B2/ja active Active
-
2023
- 2023-12-12 JP JP2023209201A patent/JP2024037850A/ja active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000502901A (ja) * | 1995-12-30 | 2000-03-14 | デルタ、バイオテクノロジー、リミテッド | 成長ホルモンおよび血清アルブミンに対する組換え融合タンパク質 |
JP2005514060A (ja) * | 2001-12-21 | 2005-05-19 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合タンパク質 |
JP2007522806A (ja) * | 2004-02-09 | 2007-08-16 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合蛋白質 |
JP2008043285A (ja) * | 2006-08-18 | 2008-02-28 | Nipro Corp | 糖鎖含有アルブミン、その製造方法およびその用途 |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017159540A1 (ja) * | 2016-03-14 | 2017-09-21 | Jcrファーマ株式会社 | 血清アルブミン-20k成長ホルモン融合タンパク質 |
US10968267B2 (en) | 2016-03-14 | 2021-04-06 | Jcr Pharmaceuticals Co., Ltd. | Serum albumin-20K growth hormone fusion protein |
EP3811962A4 (en) * | 2018-06-25 | 2022-03-16 | JCR Pharmaceuticals Co., Ltd. | PROTEIN AQUEOUS LIQUID FORMULATION |
US11738068B2 (en) | 2018-06-25 | 2023-08-29 | Jcr Pharmaceuticals Co., Ltd. | Protein-containing aqueous liquid formulation |
WO2021075526A1 (ja) * | 2019-10-17 | 2021-04-22 | Jcrファーマ株式会社 | 血清アルブミンと成長ホルモンの融合蛋白質の製造方法 |
WO2024080305A1 (ja) * | 2022-10-11 | 2024-04-18 | Jcrファーマ株式会社 | 血清アルブミンと生理活性を有する蛋白質との融合蛋白質 |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7403595B2 (ja) | 新規なヒト血清アルブミン変異体 | |
WO2018038243A1 (ja) | 抗体融合蛋白質の製造方法 | |
JP2000507456A (ja) | 拮抗的特性を有する血管内皮細胞増殖因子の変異体 | |
US10968267B2 (en) | Serum albumin-20K growth hormone fusion protein | |
WO2021085518A1 (ja) | 血清アルブミンと成長ホルモンの融合蛋白質を含有する水性医薬組成物 | |
JP2003514532A (ja) | 生成物の産生量を最適化するための核酸構築体 | |
JP2017502005A (ja) | ペグ化タンパク質の組成物を調製するための方法 | |
JP2022519061A (ja) | 副甲状腺ホルモンバリアント |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16844430 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2017539213 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2996672 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15758199 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2016319540 Country of ref document: AU Date of ref document: 20160908 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20187009487 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2016844430 Country of ref document: EP |