WO2004005335A2 - Hormone de croissance multimerisee - Google Patents
Hormone de croissance multimerisee Download PDFInfo
- Publication number
- WO2004005335A2 WO2004005335A2 PCT/DK2003/000428 DK0300428W WO2004005335A2 WO 2004005335 A2 WO2004005335 A2 WO 2004005335A2 DK 0300428 W DK0300428 W DK 0300428W WO 2004005335 A2 WO2004005335 A2 WO 2004005335A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- growth hormone
- fusion protein
- hgh
- seq
- domain
- Prior art date
Links
- 102000018997 Growth Hormone Human genes 0.000 title claims abstract description 96
- 108010051696 Growth Hormone Proteins 0.000 title claims abstract description 95
- 239000000122 growth hormone Substances 0.000 title claims abstract description 93
- 102000037865 fusion proteins Human genes 0.000 title claims description 79
- 108020001507 fusion proteins Proteins 0.000 title claims description 79
- 102100024554 Tetranectin Human genes 0.000 claims abstract description 14
- 108010013645 tetranectin Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 81
- 108090000623 proteins and genes Proteins 0.000 claims description 81
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 57
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 57
- 239000000854 Human Growth Hormone Substances 0.000 claims description 57
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 35
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 32
- 229920001184 polypeptide Polymers 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 24
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 18
- 241001465754 Metazoa Species 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 10
- 239000013598 vector Substances 0.000 claims description 9
- 108010006025 bovine growth hormone Proteins 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 6
- 208000035475 disorder Diseases 0.000 claims description 5
- 208000026928 Turner syndrome Diseases 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 150000007523 nucleic acids Chemical group 0.000 claims description 3
- 206010056438 Growth hormone deficiency Diseases 0.000 claims description 2
- 208000030159 metabolic disease Diseases 0.000 claims description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 claims 1
- 230000004071 biological effect Effects 0.000 abstract description 17
- 239000000539 dimer Substances 0.000 abstract description 12
- 239000013638 trimer Substances 0.000 abstract description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 123
- 235000018102 proteins Nutrition 0.000 description 79
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 72
- 239000011780 sodium chloride Substances 0.000 description 62
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 60
- 239000000872 buffer Substances 0.000 description 46
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 38
- 229920000936 Agarose Polymers 0.000 description 30
- 239000004202 carbamide Substances 0.000 description 30
- 210000004027 cell Anatomy 0.000 description 25
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 229920005654 Sephadex Polymers 0.000 description 18
- 239000012507 Sephadex™ Substances 0.000 description 18
- 229940121366 growth hormone derivative Drugs 0.000 description 17
- 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 14
- HNDVDQJCIGZPNO-UHFFFAOYSA-N Histidine Chemical compound OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 14
- 229930195725 Mannitol Natural products 0.000 description 14
- 238000003776 cleavage reaction Methods 0.000 description 14
- 239000013604 expression vector Substances 0.000 description 14
- 239000000594 mannitol Substances 0.000 description 14
- 235000010355 mannitol Nutrition 0.000 description 14
- 230000007017 scission Effects 0.000 description 14
- 241000700159 Rattus Species 0.000 description 13
- 235000019750 Crude protein Nutrition 0.000 description 12
- 239000012614 Q-Sepharose Substances 0.000 description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 12
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 12
- 238000005342 ion exchange Methods 0.000 description 12
- 229960004532 somatropin Drugs 0.000 description 12
- 125000000539 amino acid group Chemical group 0.000 description 11
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 230000004927 fusion Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000003155 DNA primer Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 206010062767 Hypophysitis Diseases 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 125000004122 cyclic group Chemical group 0.000 description 7
- 238000002523 gelfiltration Methods 0.000 description 7
- 239000003111 growth hormone derivative Substances 0.000 description 7
- 238000010561 standard procedure Methods 0.000 description 7
- 229910001868 water Inorganic materials 0.000 description 7
- 230000004584 weight gain Effects 0.000 description 7
- 235000019786 weight gain Nutrition 0.000 description 7
- LHOLVWOLWSNGKW-DMTCNVIQSA-N (2r,3r)-3,4-bis(sulfanyl)butane-1,2-diol Chemical compound OC[C@@H](O)[C@@H](S)CS LHOLVWOLWSNGKW-DMTCNVIQSA-N 0.000 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 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 241000672609 Escherichia coli BL21 Species 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- LZCZIHQBSCVGRD-UHFFFAOYSA-N benzenecarboximidamide;hydron;chloride Chemical compound [Cl-].NC(=[NH2+])C1=CC=CC=C1 LZCZIHQBSCVGRD-UHFFFAOYSA-N 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 238000004255 ion exchange chromatography Methods 0.000 description 6
- 108700010833 lambda phage proteins Proteins 0.000 description 6
- 239000012160 loading buffer Substances 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- 230000003204 osmotic effect Effects 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 210000003635 pituitary gland Anatomy 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 235000019419 proteases Nutrition 0.000 description 6
- 230000035939 shock Effects 0.000 description 6
- 241001515965 unidentified phage Species 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 101100217502 Caenorhabditis elegans lgg-3 gene Proteins 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 108090000909 Collectins Proteins 0.000 description 3
- 102000004405 Collectins Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000006471 dimerization reaction Methods 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 238000009578 growth hormone therapy Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229940065770 humatrope Drugs 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 108700041430 link Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- NBWRJAOOMGASJP-UHFFFAOYSA-N 2-(3,5-diphenyl-1h-tetrazol-1-ium-2-yl)-4,5-dimethyl-1,3-thiazole;bromide Chemical compound [Br-].S1C(C)=C(C)N=C1N1N(C=2C=CC=CC=2)N=C(C=2C=CC=CC=2)[NH2+]1 NBWRJAOOMGASJP-UHFFFAOYSA-N 0.000 description 1
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 206010013883 Dwarfism Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 1
- 101000664737 Homo sapiens Somatotropin Proteins 0.000 description 1
- 101000997832 Homo sapiens Tyrosine-protein kinase JAK2 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains 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
- 229930182816 L-glutamine Natural products 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010068542 Somatotropin Receptors Proteins 0.000 description 1
- 101800001707 Spacer peptide Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 239000012911 assay medium Substances 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
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000000960 hypophysis hormone Substances 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4726—Lectins
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
Definitions
- the present invention relates to multimerised growth hormone polypeptides with increased biological activity.
- hGH Human growth hormone
- somatotropin or somatropin Human growth hormone
- PA 1640 AMP Humatrope Prescribing Information
- An alternative route of administration is subcutaneous or intramuscular injection.
- This route offers slower absorption from the site of administration, thus causing a sustained release effect and thereby an enhanced plasma half-life of 3.8 and 4.9 hours for subcutaneous and intramuscular injection, respectively (Eli Lilly, Humatrope Prescribing Information, PA 1640 AMP).
- PA 1640 AMP Humatrope Prescribing Information
- significantly lower plasma levels are achieved and, thus, a similar frequency of injection, as is required with intravenous administration, may be necessary to produce a comparable therapeutic effect.
- a regimen of repeated injections may be highly inconvenient, or even impracticable, for the patients in need of hGH therapy.
- there is a need for alternative growth hormone products which can offer a more suitable administration schedule as compared to the existing short-acting growth hormone therapies.
- hGH Human growth hormone
- This monomeric 22,000-dalton pituitary hormone consists of a single chain of 191 amino acid residues and is cross-linked by two disulfide bridges. It exhibits a multitude of biological effects derived from the interaction between hGH and its cell surface receptor (hGHR), which is a single membrane-spanning type I glycoprotein.
- hGHR cell surface receptor
- hGH causes the activation of the hGHR-associated cytoplasmic tyrosine kinase, JAK2.
- hGH preparations have been prepared from human pituitaries, but presently the products on the market are produced by recombinant methods. hGH is primarily used to stimulate linear growth in patients with hypopituitary dwarfism or Turner's syndrome. Growth hormone therapy is also presently used in children to promote growth and in adults to improve muscle strength, reduce fat mass and improve metabolic profiles, which could predispose to cardiovascular disease.
- hGH human growth hormone
- bGH bovine growth hormone
- EDC cross-linking reagent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide
- hGH and bGH was randomly derivatized to give predominantly amide-linked dimers but also amide-linked multimers, depending on the concentration of EDC reagent used.
- the final protein preparation was heterogeneous due to non-specific reaction of the EDC reagent with various amino acids in the protein, including lysine, aspartic acid and glutamic acid residues and the amino- and carboxy-termini. It was found that there was a correlation between the amount of GH dimer present in the heterogeneous protein preparation and biological activity. Clearly, an injection of such a heterogeneous preparation into humans would be undesirable due to the toxic nature of EDC and the potential immunogenic response to the unnatural amide bond formed between the proteins. Generating consistent batches of a purified protein also would be difficult at the manufacturing scale.
- WO 01/79480 discloses that the stability, and hence the shelf-life, of hGH may be increased by the fusion of one hGH-molecule to a human serum albumin (HA) molecule which comprises a specific amino acid sequence. It is suggested that the increased stability results in increased biological activity of the HA-hGH fusion protein.
- HA human serum albumin
- the fusion of at least one growth hormone polypetide molecule to a multimerisation domain, and thereby the provision of growth hormone multimers, e.g. dimers and trimers, may significantly increase the biological activity, including plasma half life, of growth hormones in general and human growth hormone in particular.
- growth hormone multimers e.g. dimers and trimers
- alternative growth hormone molecules which may have increased plasma half- life. This increased half life will result in a more suitable administration schedule as compared to the existing short-acting growth hormone therapies.
- the invention relates in a first aspect to a growth hormone fusion protein comprising at least one growth hormone and a multimerisation domain.
- polypeptide complex comprising at least two fusion proteins according to the invention.
- the present invention also provides a composition comprising the growth hormone fusion protein or the polypeptide complex according to the invention.
- the invention pertains to a method of treating a disease or disorder in a mammal comprising the step of administering the growth hormone fusion protein or the polypeptide complex according to the invention to a mammal, and the use of the growth hormone fusion protein or the polypeptide complex for the preparation of a pharmaceutical composition.
- a kit comprising the composition according to the invention, and a method of producing the growth hormone fusion protein and the polypeptide complex according to the invention.
- the primary objective of the present invention is to provide alternative growth hormone molecules which are growth hormone fusion proteins comprising at least one growth hormone and a multimerisation domain providing for growth hormone multimers.
- the fusion of at least one growth hormone polypeptide to a multimerisation domain provides for growth hormone multimers, e.g. growth hormone dimers and growth hormone trimers, having significantly increased biological activity as compared to native growth hormone, such as human growth hormone.
- increased biological activity includes prolonged plasma half-life, i.e. a longer circulating half-life relative to native monomeric growth hormone. It will be appreciated that by “half-life” or “plasma half-life” is meant the time for a drug concentration in the plasma to be reduced by one-half, typically measured after administration of a selected dose. By “plasma” herein is meant either plasma or serum.
- the plasma half-life of the growth hormone multimers according to the invention is preferably increased compared to that of native monomeric growth hormone.
- the plasma half-life is increased by at least 5 %, such as at least 10 %, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 40% such as at least 50%, for example at least 75%, such as at least 100%.
- the plasma half life is increased at least about 3, 4, 5, 6, 7, 8, 9, 10, 15 or 18 times as compared to native monomeric growth hormone.
- the, plasma half-life is increased at least about 20, 30, 40 or 50 times, as compared to native monomeric growth hormone.
- An increased plasma half-life may have profound implications for the use of the growth hormone multimers according to the invention in the treatment of various indications. It is therefore expected that the clinical effect of the growth hormone multimers according to the invention is superior to the effect of native monomeric growth hormone, such as human growth hormone.
- the growth hormone multimers according to the invention may have improved stability, and hence improved shelf-life, as compared to native monomeric growth hormone.
- Increased biological activity can also encompass a combination of the above-described activities, for example, a growth hormone fusion protein or growth hormone multimer with higher potency that also exhibits a prolonged circulating half-life and improved stability. Because the growth hormone fusion proteins of the present invention may have increased biological activity, it is contemplated that the frequency with which they must be administered may be reduced, or the amount administered to achieve an effective dose can be reduced.
- the growth hormone fusion proteins according to the invention may provide for growth hormone multimers having increased affinity for growth hormone receptors. Such an increased affinity can result in an increased stimulation of the signal generated by the activation of the receptors. This may imply that a reduced quantity of growth hormone fusion proteins would then be necessary over the course of treatment, as compared to the quantity necessary if native growth hormone was used.
- growth hormone or "GH” is intended to refer to either natural or recombinant pituitary growth hormone, regardless of the source. The term is limited only in that the material must demonstrate pituitary growth hormone biological activity in a recipient such as a human.
- hGH human growth hormone
- somatotropin or somatropin recombinant growth hormone
- rGH recombinant growth hormone
- bGH bovine growth hormone
- pGH porcine growth hormone
- the growth hormone is linked to a multimerisation domain.
- multimerisation domain is a peptide, a protein or part of a protein which is capable of interacting with other, similar or identical multimerisation domains.
- the interaction is of the type that produces multimeric proteins or polypeptides. Such an interaction may be caused by covalent bonds between the components of the multimerisation domains as well as by hydrogen bond forces, hydrophobic forces, van derWaals forces and salt bridges.
- the multimerisation domain peptide is a dimerising domain, a trimerising domain, a tetramerising domain, a pentamerising domain or a hexamerising domain.
- WO 95/31540 describes polypeptides comprising a collectin neck region.
- the amino acid sequence constituting the collectin neck region may be attached to any polypeptide of choice. Trimers can then be made under appropriate conditions with three polypeptides comprising the collectin neck region amino acid sequence.
- the multimerisation domain of the fusion protein according to the invention may comprise coiled-coil dimerization domains such as leucine zipper domains which are found in certain DNA-binding polypeptides.
- the multimerisation domain according to the invention may also comprise a dimerization domain which is an immunoglobulin Fab constant domain, such as an immunoglobulin heavy chain CH1 constant region or an immunoglobulin light chain constant region.
- the multimerisation domain is derived from tetranectin, and more specifically comprises the tetranectin trimerising structural element (hereafter termed TTSE) which is described in detail in WO 98/56906.
- TTSE tetranectin trimerising structural element
- SEQ ID NO 17 The amino acid sequence of TTSE is shown in SEQ ID NO 17.
- the trimerising effect of TTSE is caused by a coiled coil structure which interacts with the coiled coil structure of two other TTSEs to form a triple alpha helical coiled coil trimer which is exceptionally stable even at relatively high temperatures.
- TTSE is also intended to embrace variants of a TTSE of a naturally occurring member of the tetranectin family of proteins, variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the capability of the TTSE to form alpha helical coiled coil trimers.
- the fusion protein according to the invention may comprise a TTSE as a multimerisation domain, which comprises a sequence having at least 68% amino acid sequence identity with the sequence of SEQ ID NO SEQ ID NO 17, such as at least 75%, including at least 87%, such as at least 92%.
- TTSE 50 of the TTSE may advantageously be mutagenised to serine, threonine, methionine or to any other amino acid residue in order to avoid formation of an unwanted inter-chain disulphide bridge, which could lead to unwanted multimerisation.
- the TTSE multimerisation domain may e.g. be modified by (i) the incorporation of polyhistidine sequence and/or a cleavage site for the Blood Coagulating Factor X a , (ii) replacing Cys 50 with Ser, and (iii) by including a C- terminal KGS sequence.
- An example of such a modified TTSE is given as SEQ ID NO 18, and is designated TripA.
- the TTSE multimerisation domain may be modified by truncating the amino acid sequence and removing the C-terminal amino acid residues C50 and L51. This modified TTSE is in the present context designated TripD.
- the growth hormone may either be linked to the N- or the C-terminal amino acid residue of the multimerisation domain.
- it is also envisaged that in certain embodiments it may be advantageous to link a growth hormone to both the N-terminal and the C-terminal of the multimerisation domain of the fusion protein, and thereby providing a fusion protein comprising two growth hormones.
- a flexible molecular linker optionally may be interposed between, and covalently join, the growth hormone and the multimerisation domain.
- the linker is a polypeptide sequence of about 1-20 amino acid residues, such as about 2-10 amino acid residues, including 3-7 amino acid residues.
- the linker is essentially non-immunogenic, not prone to proteolytic cleavage and does not comprise amino acid residues which are known to interact with other residues (e.g. cystein residues).
- Preferred examples of spacer or linker peptides include those, which have been used to link proteins without substantially impairing the function of the linked proteins or at least without substantially impairing the function of one of the linked proteins. More preferably the linkers or spacers have been used to link proteins comprising coiled-coil structures.
- linker sequences which are believed to be suitable for linking growth hormone to the multimerisation domain of the present invention.
- Tetranectin based linker may include the tetranectin amino acid residues 53- 56, which in tetranectin forms a beta-strand, and the residues 57-59 which forms a turn in tetranectin (Nielsen et al., 1997).
- the sequence of the segment is GTKVHMK.
- This linker has the advantage that it in native tetranectin is bridging the trimerisation domain with the CRD-domain, and hence it is contemplated to be well suited for connecting the trimerisation domain to another domain in general. Furthermore, the resulting construct is not expected to be more immunogenic than the construct without a linker.
- the tetranectin based linker is highly preferred when the multimerisation domain is TTSE.
- Fibronectin based linker The linker may be chosen as a sub-sequence from the connecting strand 3 from human fibronectin, this corresponds to amino acid residues 1992-2102 (SWISS-PROT numbering, entry P02751).
- the subsequence: PGTSGQQPSVGQQ covering amino acid residues number 2037-2049 is used, and within that subsequence the segment GTSGQ corresponding to amino acid residues 2038-2042 is more preferable.
- This construct has the advantage that it is know not to be highly prone to proteolytic cleavage and is not expected to be highly immunogenic bearing in mind that fibronectin is present at high concentrations in plasma.
- Human lgG3 upper hinge based linker The 10 amino acid residue sequence derived from the upper hinge region of murine lgG3, PKPSTPPGSS, has been used for the production of antibodies dimerised trough a coiled coil (Pack et al., 1992) and may be useful as a spacer peptide according to the present invention. Even more preferable may be a corresponding sequence from the upper hinge region of human lgG3. Sequences from human lgG3 are not expected to be immunogenic in humans.
- flexible amino acid linker sequences include SGGTSGSTSGTGST, AGSSTGSSTGPGSTT or GGSGGAP. These sequences have previously been used for the linking of designed coiled coils to other protein domains (Mutter et al., 2000).
- the linker interposed between the growth hormone and the multimerisation domain may be the amino acid sequences GSA or GSQEGSA.
- the growth hormone fusion protein according to the invention is TripA-GSA-hGH (SEQ ID NO 4), MOTripA-GSA-hGH (SEQ ID NO 6), TripA-GSQEGSA- hGH (SEQ ID NO 9), MOTripA-GSQEGSA-hGH (SEQ ID NO 11), TripA-hGH (SEQ ID NO 16) or TripD-GSQEGSA-hGH (SEQ ID NO 23).
- the growth hormone fusion proteins and the multimer polypeptide complex according of the present invention may be expressed in any suitable standard protein expression system by providing a host cell capable of expressing the fusion proteins and/or the multimer polypeptide complex in recoverable amounts.
- the expression systems are systems from which the desired protein may readily be isolated and refolded in vitro.
- prokaryotic expression systems are preferred since high yields of protein can be obtained and efficient purification and refolding strategies are available.
- the primary amino acid sequence for the growth hormone fusion proteins of the present invention is chosen, one of ordinary skill in the art can easily design appropriate recombinant DNA constructs which will encode the desired proteins, taking into consideration such factors as codon biases in the chosen host, the need for secretion signal sequences in the host, the introduction of proteinase cleavage sites within the signal sequence, and the like.
- These recombinant DNA constructs may be inserted in-frame into any of a number of expression vectors appropriate to the chosen host.
- the choice of an appropriate or favourite expression vector is, again, a matter well within the ability and discretion of the skilled practitioner.
- the expression vector will include a strong promoter to drive expression of the recombinant constructs.
- the growth hormone fusion proteins and the polypeptide complex may be produced by a method which comprises the steps of (i) providing a recombinant vector comprising the isolated nucleic acid sequence encoding the fusion protein of the invention which is, optionally, operatively linked to a promotor, (ii) transforming a host cell with this recombinant vector, (iii) culturing the host cell under conditions to express the fusion protein, and (iv) optionally isolating the growth hormone fusion protein and/or polypeptide complex.
- multimers of the growth hormone are formed under appropriate conditions resulting in a multimeric polypeptide complex.
- growth hormone dimers, trimers, tretramers, pentamers, hexamers or even higher -mers can be prepared depending on the type of multimerisation domain being linked to the growth hormone.
- a multimeric polypeptide complex comprising at least two fusion proteins, such as at least three, including at least four, such at least five, including at least six fusion proteins.
- the presence of a growth hormone multimer, such as a growth hormone trimer may be ascertained by well known techniques such as gelfiltration, SDS-PAGE, or native SDS gel electrophoresis depending on the nature of the multimer.
- the growth hormone fusion protein in accordance with the invention may be used for the preparation of a pharmaceutical composition by any suitable method well known in the art.
- the composition may together with the growth hormone fusion protein, comprise one or more acceptable carriers therefore, and optionally other therapeutic ingredients.
- the carriers must be acceptable in the sense of being compatible with the other ingredients and not deleterious to the recipient thereof.
- methods for the preparation of pharmaceutical compositions include the step of bringing into association the active ingredient and a carrier.
- the therapeutic application of the present invention comprises the treatment of a disease or disorder in an animal, by administering a therapeutically effective amount of the growth hormone fusion protein or the polypeptide complex according to the invention to the animal in need thereof.
- Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular animal or patient undergoing treatment.
- it can be necessary to provide for repeated administration i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
- the therapeutically effective amount or dosage is in the range from at least about 0.001 to 500 milligrams per kilogram of animal, and preferably from at least about 0.1 to 100 milligrams per kilogram of animal per single or multiple administration, depending upon the specific activity of contained in the composition.
- Preferred doses can optionally include 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500
- treatment of animals can be provided as a onetime or periodic dosage of the growth hormone fusion protein or the polypeptide complex of the present invention at an amount of 0.1 to 100 mg/kg per day, such as 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83,
- the disease or disorder is growth hormone deficiency, turner syndrome and metabolic disorder.
- the growth hormone fusion protein or the polypeptide complex according to the present invention may be directly administered to the animal by any suitable technique, including parenterally, and can be administered locally or systemically.
- the specific route of administration depends, e.g., on the medical history of the animal.
- parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration.
- the growth hormone fusion protein or the polypeptide complex according to the invention can be formulated as a solution, suspension, emulsion or lyophilised powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle.
- a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
- the vehicle or lyophilised powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
- the formulation is sterilized by known or suitable techniques.
- the animals potentially treatable by the growth hormone fusion protein or the polypeptide complex herein include mammals such as bovine, ovine, and porcine animals.
- mammals such as bovine, ovine, and porcine animals.
- the preferred mammal herein is a human.
- the growth hormone fusion protein or the polypeptide complex herein may be used for stimulating linear growth in human pediatric patients who lack adequate normal endogenous growth hormone and patients with Turner syndrome.
- Trimeric growth hormone derivative TripA-GSA-hGH
- the Expression vector pT76HFXTripA-GSA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHN: 5- GCT CAC GGG ATC CGC TTT CCC AAC CAT TCC CTT AT-3 [SEQ ID NO:1] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind 111 cut E.
- TripA-GSA-hGH The nucleotide sequence of the resulting TripA-GSA-hGH, is given as SEQ ID NO:3 and the amino acid sequence encoded by the TripA-GSA-hGH insert is given in SEQ ID NO:4.
- the recombinant human Growth Hormone derivative TripA-GSA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-GSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986).
- the trimeric fusion protein, TripA-GSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5mM 2- mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded on the Ni2+NTA-agarose column using the cyclic refolding procedure described by Th ⁇ gersen et al. (International Patent Application W09418227) with a gradient manager profile as described below in Table 1 and 0.5 M NaCl, 50 mM Tris-HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B. TABLE 1
- Step Time 1 Flow %A ' %B Step Tine Fiow %A %B
- TripA-GSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.
- the TripA-GSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G- 25, and the protein was then applied onto a Q-Sepharose (Amersham Biosciences) ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.
- the monomeric fraction eluted from the ion exchange column was gelfiltrated in to a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa is inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8.
- the Expression vector pT76HFXI10TripA-GSA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHN: 5- GCT CAC GGG ATC CGC TTT CCC AAC CAT TCC CTT AT-3 [SEQ ID NO:1] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut E. coli expression vector, pT76HFXI10Tripa using standard procedures.
- the resulting nucleotide sequence of the MOTripA-GSA-hGH insert is given as SEQ ID NO:5.
- the amino acid sequence encoded by the MOTripA-GSA-hGH insert is given in SEQ ID NO:6.
- the recombinant human growth hormone derivative MOTripA-GSA-hGH was produced by growing and expressing the plasmid pT76HFXI10TripA-GSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier and Moffat, J. Mol. Biol., 189: 113-130, 1986. Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation.
- Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base). Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation. The protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- the fusion protein, MOTripA-GSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8, and 5 mM reduced gluthatione as buffer B.
- the HOTripA-GSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.
- the HOTripA-GSA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose (Amersham Biosciences) ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.
- the ion exchange protein was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8.
- Trimeric growth hormone derivative TripA-GSQEGSA-hGH
- the Expression vector pT76HFXTripA-GSQEGSA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXtripa (Lorentsen et al.
- the nucleotide sequence of the TripA-GSQEGSA-hGH insert is given as SEQ ID NO:8 and the amino acid sequence encoded by the TripA-GSQEGSA-hGH is given as SEQ ID NO:9.
- the recombinant human Growth Hormone TripA-GSQEGSA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-GSQEGSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE6 at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).
- Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation.
- the protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- a buffer containing 6 M guanidinium chloride 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).
- the fusion protein, TripA-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.
- the TripA-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 20 mM EDTA pH 8.
- the TripA-GSQEGSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 400 mM NaCl and 50 mM Tris- HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.
- the monomeric fraction eluted from the ion exchange column was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8.
- the Expression vector pT76HFXI10TripA-GSQEGSA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXI10tripa using standard procedures.
- the nucleotide sequence of MOTripA- GSQEGSA-hGH insert is given as SEQ ID NO: 10.
- the amino acid sequence encoded by the MOTripA-GSQEGSA-hGH insert is
- the recombinant human Growth Hormone HOTripA-GSQEGSA-hGH was produced by growing and expressing the plasmid pT76HFXI10TripA-GSQEGSA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE ⁇ at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).
- Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation.
- the protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- a buffer containing 6 M guanidinium chloride 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).
- the fusion protein, MOTripA-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2- mercaptoethanol, until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.
- the MOTripA-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 10 mM EDTA pH 8.
- the MOTripA-GSQEGSA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers ( due to inter-hGHdomain disulfide bridge formation) eluted later.
- the ion exchange protein was gelfiltrated in to a buffer contaning 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 O and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8. Fractions containing the cleaved purified recombinant protein was gelfiltrated into PBS mannitol buffer (220mM mannitol, 3.24mM histidin and 32mM phenol) buffer on a Sephadex G-25 column.
- the DNA fragment TripA-hGH (SEQ ID NO: 12) was amplified from a so-called assembly reactions combining the two DNA fragments BamHI-TripA and hGH-Hind.
- the BamHI-TripA fragment was amplified from pT76HFXtripa vector (with the oligonucleotide primers AssFXTripAN: 5-GCT CGA CGG ATC CAT CCA GGG AAG AGG TGA GCC ACC AAC CCA GAA GC-3 [SEQ ID NO: 13] and AssTripAC 5-GGT TGG GAA CTT CAG GGA GAC CGT GTG C-3 [SEQ ID NO:14 ]), and the hGH-Hind fragment was amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers AsshGHN: 5-CTC CCT GAA GTT CCC AAC CAT TCC CTT ATC C-3
- the Expression vector pT76HFXTripA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment Ass-TripA-hGH (SEQ ID NO:12), into a BamH I and Hind III cut E. coli expression vector, pT76H (Christensen et al., 1991) using standard procedures.
- the resulting nucleotide sequence of the TripA-hGH insert is given as SEQ ID NO:12
- the amino acid sequence encoded by the TripA-hGH insert is given as SEQ ID NO:16.
- the recombinant human Growth Hormone derivative TripA-hGH was produced by growing and expressing the plasmid pT76HFXTripA-hGH in E. coli BL21 cells in a medium scale (2x1 litre) as described by Studier et al. (1986). Exponentially growing cultures at 37°C were at OD600 0.8 infected with bacteriophage lambdaCE ⁇ at a multiplicity of approximately 5. Cultures were grown at 37°C for another three hours before cells were harvested by centrifugation. Cells were lysed by osmotic shock and sonification and total cellular protein extracted into phenol (adjusted to pH 8 with Trisma base).
- Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation.
- the protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- a buffer containing 6 M guanidinium chloride 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose).
- the fusion protein, TripA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1 mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.
- TripA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50mM Tris-HCl pH 8 and 10 mM EDTA pH 8.
- the TripA-hGH protein was gelfiltrated into a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein was eluted over 5 column volumes with a linear gradient from 8 M urea, 5 mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 500 mM NaCl and 50 mM Tris-HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.
- the monomeric fraction eluted from the ion exchange column was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein were eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8.
- Fractions containing the cleaved purified recombinant protein TripA-hGH was gelfiltrated into mannitol buffer (220mM mannitol, 3.24mM histidin and 32mM phenol) buffer on a Sephadex G-25 column.
- Trimeric growth hormone derivative TripD-GSQEGSA-hGH
- the Expression vector pT76HFXTtipd was constructed by ligation of the Bgl II and Hind III restricted DNA fragment FXtripd (SEQ ID NO:19) amplified from the expression vector pT76HFXtripa (Lorentsen et al., 2000) with the oligonucleotide primers N- pT7H6FXGTripA (SEQ ID NO:20) and C-pT7H6FXGTrip49 (SEQ ID NO:21 ) in to a BamH I and Hind III cut vector pT76H (Christensen et al., 1991).
- the Expression vector pT76HFXTripD-GSQEGSA-hGH was constructed by ligation of the BamH I and Hind III restricted DNA fragment amplified from cDNA, isolated from human pituitary gland (Clontech Laboratories, Inc) (with the oligonucleotide primers HGHgsqegsaN: 5- GCT CAC GGG ATC CCA GGA AGG CTC CGC TTT CCC AAC CAT TCC CTT AT -3 [SEQ ID NO: 7] and HGHC 5-GCT CCA GAA GCT TAG AAG CCA CAG CTG CCC-3 [SEQ ID NO: 2]) into a BamH I and Hind III cut vector, pT76HFXtripd using standard procedures.
- the nucleotide sequence of the TripD- GSQEGSA-hGH insert is given as SEQ ID NO:22 and the amino acid sequence encoded by the TripD-GSQEGSA-hGH is given as SEQ ID NO:23.
- Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and centrifugation.
- the protein pellet was dissolved in a buffer containing 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- a buffer containing 6 M guanidinium chloride 50 mM Tris-HCl pH 8 and 50 mM dithioerythriol.
- the crude protein preparation was applied to a Ni2+ activated NTA-agarose colum (Ni2+NTA-agarose, Qiagen).
- the fusion protein, TripD-GSQEGSA-hGH was purified from the majority of coli and lambda phage proteins by washing with one column volume of the loading buffer followed by 6 M guanidinium chloride, 50 mM Tris-HCl pH 8 and 5 mM 2-mercaptoethanol until the optical density (OD) at 280 nm of the eluate was stable.
- the fusion protein was refolded as described above on the Ni2+NTA-agarose column using a gradient manager profile as described in Table 1 and 0.5 M NaCl, 50 mM Tris- HCl pH 8, 1mM reduced gluthatione and 0.1 mM oxidized gluthatione as buffer A and 8 M urea, 0.5 NaCl, 50 mM Tris-HCl pH 8 and 5 mM reduced gluthatione as buffer B.
- TripD-GSQEGSA-hGH protein was eluted from the Ni2+NTA-agarose column with a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8 and 20 mM EDTA pH 8.
- the TripD-GSQEGSA-hGH protein was gelfiltrated in to a buffer containing 8 M urea 5 mM NaCl and 25 mM Tris-HCl pH 8 on a Sephadex G-25, and the protein was then applied onto a Q-Sepharose ion exchange column. Protein were eluted over 5 column volumes with a linear gradient from 8 M urea, 5mM NaCl and 25 mM Tris-HCl pH 8 to 8 M urea, 400 mM NaCl and 50 mM Tris- HCl pH 8. The folded protein was eluted in the beginning of the gradient, whereas dimers and higher order multimers (due to inter-hGHdomain disulfide bridge formation) eluted later.
- the monomeric fraction eluted from the ion exchange column was gelfiltrated into a buffer containing 100 mM NaCl and 25 mM Tris-HCl pH 8 and cleaved with restriction protease FXa for 5 hours at 37°C in a weight to weight ratio of approximately 100:1. FXa was inhibited after cleavage by addition of Benzamidine hydrochloride to 1 mM.
- the recombinant protein was diluted with 10 volumes of H 2 0 and then isolated from uncleaved fusion protein, FXa and the liberated fusion tail by ion exchange chromatography on a Q-Sepharose column, the protein was eluted over 5 column volumes with a linear gradient from 5 mM NaCl and 25 mM Tris-HCl to 250 mM NaCl and 50 mM Tris-HCl pH 8.
- the trimeric human growth hormone derivatives TripA-hGH, TripA-GSA-hGH, TripA- GSQEGSA-hGH, TripD-GSQEGSA-hGH were expressed, refolded, and purified as described in Example 1.
- Nb2-11 rat lymphoma cells obtained from the European Collection of Cell Cultures, ecacc
- RPMI 1640 medium supplemented with antibiotics at 1% (penicillin 100 U/mL and streptomycin 100 ⁇ g/mL), 2 mM L-glutamine, 10% horse serum and 10% fetal calf serum at standard conditions.
- the cells were transferred to "quiescent" medium consisting of RPMI 1640 medium supplemented with antibiotics at 1%, 2 mM L- glutamine, 10% horse serum and 1 % fetal calf serum 24 hours prior to assay start.
- Nb2-11 cells were transferred to "assay" medium (quiescent medium without fetal calf serum) and 2x10 4 cells were seeded into each well of a 96- well microtiter plates. Fifty microliters samples of each of the trimeric human growth hormone derivatives or controls diluted in assay medium in order to yield a dose response result, were added to each well. Plates were incubated for 48 or 72 hours at standard conditions. To measure cell proliferation MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5- di-phenyl-tetrazolium bromide) was used as part of the CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit (Promega) according to the manufacturers protocol. Experiments were done in triplicate. The results were read on a microtiter plate reader, where the amount of formazan produced, measured as OD492, reflected the level of cellular proliferation. All dilutions were made in triplicate.
- the trimeric human growth hormone derivatives TripA- hGH, TripA-GSQEGSA-hGH and TripD-GSQEGSA-hGH are capable of stimulating Nb- 2 cell proliferation significantly better or at least as good as monomeric somatropin.
- the trimeric derivative TripA-GSA-hGH stimulates Nb- 2 cell proliferation at a level which is about 50 times lower than for somatropin.
- the rats were weighed daily from the first administration of test sample and in the following nine days. The development of body weight was analyzed using standard statistical procedures calculating the mean weight gain for each group of rats relative to the mean weight at day 0.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003239777A AU2003239777A1 (en) | 2002-07-05 | 2003-06-23 | Multimerised growth hormone fusion proteins |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US39338302P | 2002-07-05 | 2002-07-05 | |
DKPA200201062 | 2002-07-05 | ||
US60/393,383 | 2002-07-05 | ||
DKPA200201062 | 2002-07-05 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2004005335A2 true WO2004005335A2 (fr) | 2004-01-15 |
WO2004005335A3 WO2004005335A3 (fr) | 2004-02-26 |
Family
ID=30116815
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK2003/000428 WO2004005335A2 (fr) | 2002-07-05 | 2003-06-23 | Hormone de croissance multimerisee |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2003239777A1 (fr) |
WO (1) | WO2004005335A2 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2727936A1 (fr) | 2006-11-22 | 2014-05-07 | Bristol-Myers Squibb Company | Agents thérapeutiques ciblés sur la base des protéines modifiées de récepteurs de tyrosine kinases, dont le IGF-IR |
EP2799448A1 (fr) | 2008-05-22 | 2014-11-05 | Bristol-Myers Squibb Company | Protéines de domaine d'échafaudage à base de fribronectine multivalente |
JP2014529602A (ja) * | 2011-08-25 | 2014-11-13 | エフ.ホフマン−ラ ロシュアーゲーF.Hoffmann−La Roche Aktiengesellschaft | 短縮されたテトラネクチン−アポリポプロテインa−i融合タンパク質、それを含む脂質粒子、及びその使用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996037621A2 (fr) * | 1995-05-23 | 1996-11-28 | Morphosys Gesellschaft Für Proteinoptimierung Mbh | Proteines multimeres |
WO1997024445A1 (fr) * | 1995-12-30 | 1997-07-10 | Delta Biotechnology Limited | Proteines de fusion recombinees d'hormone de croissance et d'albumine serique |
WO1998056906A1 (fr) * | 1997-06-11 | 1998-12-17 | Thoegersen Hans Christian | Module formant des trimeres |
WO2002048189A2 (fr) * | 2000-12-13 | 2002-06-20 | Borean Pharma A/S | Échantillothèques combinatoires de protéines présentant la structure en échafaudage des domaines lectonoïdes de type c |
WO2003054152A2 (fr) * | 2001-12-10 | 2003-07-03 | Nuvelo, Inc. | Nouveaux acides nucleiques et polypeptides |
-
2003
- 2003-06-23 WO PCT/DK2003/000428 patent/WO2004005335A2/fr active Application Filing
- 2003-06-23 AU AU2003239777A patent/AU2003239777A1/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996037621A2 (fr) * | 1995-05-23 | 1996-11-28 | Morphosys Gesellschaft Für Proteinoptimierung Mbh | Proteines multimeres |
WO1997024445A1 (fr) * | 1995-12-30 | 1997-07-10 | Delta Biotechnology Limited | Proteines de fusion recombinees d'hormone de croissance et d'albumine serique |
WO1998056906A1 (fr) * | 1997-06-11 | 1998-12-17 | Thoegersen Hans Christian | Module formant des trimeres |
WO2002048189A2 (fr) * | 2000-12-13 | 2002-06-20 | Borean Pharma A/S | Échantillothèques combinatoires de protéines présentant la structure en échafaudage des domaines lectonoïdes de type c |
WO2003054152A2 (fr) * | 2001-12-10 | 2003-07-03 | Nuvelo, Inc. | Nouveaux acides nucleiques et polypeptides |
Non-Patent Citations (4)
Title |
---|
JEREMY THORNER ET AL: "Applicatins of Chimeric Genes and Hybrid Proteins" METHODS IN ENZYMOLOGY, vol. 328, 2000, pages 260-283, XP002260661 ISSN: 0076-6879 * |
LORENTSEN R H ET AL: "The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain." THE BIOCHEMICAL JOURNAL. ENGLAND 1 APR 2000, vol. 347 Pt 1, 1 April 2000 (2000-04-01), pages 83-87, XP002260660 ISSN: 0264-6021 * |
MICHEL JAQUINOD ET AL: "Mass Spectrometric Characterisation of Post-Translational Modification and Genetic Variation in Human Tetranectin" BIOL. CHEM., vol. 380, November 1999 (1999-11), pages 1307-1314, XP002260659 * |
NIELSEN B B ET AL: "Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil." FEBS LETTERS. NETHERLANDS 28 JUL 1997, vol. 412, no. 2, 28 July 1997 (1997-07-28), pages 388-396, XP002260662 ISSN: 0014-5793 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2727936A1 (fr) | 2006-11-22 | 2014-05-07 | Bristol-Myers Squibb Company | Agents thérapeutiques ciblés sur la base des protéines modifiées de récepteurs de tyrosine kinases, dont le IGF-IR |
EP3156415A1 (fr) | 2006-11-22 | 2017-04-19 | Bristol-Myers Squibb Company | Agents thérapeutiques ciblés sur la base des protéines modifiées de récepteurs de tyrosine kinases, dont le igf-ir |
EP2799448A1 (fr) | 2008-05-22 | 2014-11-05 | Bristol-Myers Squibb Company | Protéines de domaine d'échafaudage à base de fribronectine multivalente |
JP2014529602A (ja) * | 2011-08-25 | 2014-11-13 | エフ.ホフマン−ラ ロシュアーゲーF.Hoffmann−La Roche Aktiengesellschaft | 短縮されたテトラネクチン−アポリポプロテインa−i融合タンパク質、それを含む脂質粒子、及びその使用 |
JP2017171670A (ja) * | 2011-08-25 | 2017-09-28 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | 短縮されたテトラネクチン−アポリポプロテインa−i融合タンパク質、それを含む脂質粒子、及びその使用 |
Also Published As
Publication number | Publication date |
---|---|
WO2004005335A3 (fr) | 2004-02-26 |
AU2003239777A8 (en) | 2004-01-23 |
AU2003239777A1 (en) | 2004-01-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230265143A1 (en) | Recombinant human epo-fc-fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo | |
KR101651977B1 (ko) | 장기 작용 폴리펩티드 및 그의 생산 및 투여 방법 | |
EP1833847B1 (fr) | Polypeptides de fusion contenant igf-1 et utilisations therapeutiques de ces polypeptides | |
JP7174149B2 (ja) | GLP1-Fc融合タンパク質及びその複合体 | |
WO1994012219A2 (fr) | Facteur de croissance insulinoide modifie | |
JP2010535781A (ja) | 肥満に対する処置 | |
WO1995032003A1 (fr) | Facteurs de croissance proches de l'insuline modifies | |
KR101304081B1 (ko) | 수식된 에리스로포이에틴 | |
WO2004005335A2 (fr) | Hormone de croissance multimerisee | |
CN115819611B (zh) | 生长激素融合蛋白及其制备方法和用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |
|
122 | Ep: pct application non-entry in european phase |