CA3032995A1 - Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same - Google Patents
Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same Download PDFInfo
- Publication number
- CA3032995A1 CA3032995A1 CA3032995A CA3032995A CA3032995A1 CA 3032995 A1 CA3032995 A1 CA 3032995A1 CA 3032995 A CA3032995 A CA 3032995A CA 3032995 A CA3032995 A CA 3032995A CA 3032995 A1 CA3032995 A1 CA 3032995A1
- Authority
- CA
- Canada
- Prior art keywords
- sst
- alb
- fusion protein
- somatostatin
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 148
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 135
- 102000009027 Albumins Human genes 0.000 title claims abstract description 37
- 108010088751 Albumins Proteins 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000000203 mixture Substances 0.000 title description 9
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical group 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 abstract description 104
- 108010056088 Somatostatin Proteins 0.000 claims abstract description 77
- 102000005157 Somatostatin Human genes 0.000 claims abstract description 76
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 74
- 229960000553 somatostatin Drugs 0.000 claims abstract description 72
- GGYTXJNZMFRSLX-DFTNLTQTSA-N somatostatin-28 Chemical compound N([C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H]1C(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)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CSSC1)C(O)=O)[C@@H](C)O)[C@@H](C)O)=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CO GGYTXJNZMFRSLX-DFTNLTQTSA-N 0.000 claims abstract description 34
- 150000001413 amino acids Chemical group 0.000 claims abstract description 13
- VVEJUSYNERNRME-XGFVQVCISA-N (4r,7s,10r,13s,16r,19s,22r,25s,28r,31s)-13,28-bis(4-aminobutyl)-25-(2-amino-2-oxoethyl)-31-[[2-[[(2s)-2-aminopropanoyl]amino]acetyl]amino]-19,22-dibenzyl-10-[(1r)-1-hydroxyethyl]-7-(hydroxymethyl)-16-(1h-indol-3-ylmethyl)-6,9,12,15,18,21,24,27,30-nonaoxo- Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=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)N1)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 VVEJUSYNERNRME-XGFVQVCISA-N 0.000 claims abstract 4
- 101500024338 Homo sapiens Somatostatin-14 Proteins 0.000 claims abstract 4
- 102400000820 Somatostatin-14 Human genes 0.000 claims abstract 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 61
- 229920001184 polypeptide Polymers 0.000 claims description 59
- 125000006850 spacer group Chemical group 0.000 claims description 38
- 239000002773 nucleotide Substances 0.000 claims description 29
- 125000003729 nucleotide group Chemical group 0.000 claims description 29
- 102100027211 Albumin Human genes 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 13
- 206010028980 Neoplasm Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 201000011510 cancer Diseases 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 102000007562 Serum Albumin Human genes 0.000 claims description 6
- 108010071390 Serum Albumin Proteins 0.000 claims description 6
- 230000002124 endocrine Effects 0.000 claims description 6
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 claims description 5
- 102100023802 Somatostatin receptor type 2 Human genes 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000035475 disorder Diseases 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 206010009944 Colon cancer Diseases 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 206010060862 Prostate cancer Diseases 0.000 claims description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 208000029559 malignant endocrine neoplasm Diseases 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 201000002120 neuroendocrine carcinoma Diseases 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 108090000586 somatostatin receptor 2 Proteins 0.000 claims description 3
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 2
- 102100029329 Somatostatin receptor type 1 Human genes 0.000 claims description 2
- 101710192647 Somatostatin receptor type 3 Proteins 0.000 claims description 2
- 102100023803 Somatostatin receptor type 3 Human genes 0.000 claims description 2
- 101710192645 Somatostatin receptor type 4 Proteins 0.000 claims description 2
- 102100023801 Somatostatin receptor type 4 Human genes 0.000 claims description 2
- 102100023806 Somatostatin receptor type 5 Human genes 0.000 claims description 2
- 108090000680 somatostatin receptor 5 Proteins 0.000 claims description 2
- 108010082379 somatostatin receptor type 1 Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 abstract description 35
- 108091006905 Human Serum Albumin Proteins 0.000 abstract description 35
- 230000027455 binding Effects 0.000 abstract description 15
- 238000001727 in vivo Methods 0.000 abstract description 11
- 101000632994 Homo sapiens Somatostatin Proteins 0.000 abstract description 9
- 238000000338 in vitro Methods 0.000 abstract description 7
- 102000045305 human SST Human genes 0.000 abstract description 5
- 230000002035 prolonged effect Effects 0.000 abstract 1
- 241000700159 Rattus Species 0.000 description 31
- 102100029563 Somatostatin Human genes 0.000 description 29
- 101800004701 Somatostatin-28 Proteins 0.000 description 29
- 210000004027 cell Anatomy 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 26
- 210000002381 plasma Anatomy 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 22
- 235000018102 proteins Nutrition 0.000 description 19
- 230000005764 inhibitory process Effects 0.000 description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 238000000746 purification Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 239000013598 vector Substances 0.000 description 11
- 229920002873 Polyethylenimine Polymers 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 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
- 239000013604 expression vector Substances 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 108050001286 Somatostatin Receptor Proteins 0.000 description 5
- 102000011096 Somatostatin receptor Human genes 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000036470 plasma concentration Effects 0.000 description 5
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 4
- 102000018997 Growth Hormone Human genes 0.000 description 4
- 108010051696 Growth Hormone Proteins 0.000 description 4
- 101000829127 Homo sapiens Somatostatin receptor type 2 Proteins 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 108010016076 Octreotide Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 108090000190 Thrombin Proteins 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000003491 cAMP production Effects 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 239000000122 growth hormone Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 229960002700 octreotide Drugs 0.000 description 4
- 229960004072 thrombin Drugs 0.000 description 4
- 238000012286 ELISA Assay Methods 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 108020001580 protein domains Proteins 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- PUDHBTGHUJUUFI-SCTWWAJVSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-n-[(2s,3r)-1-amino-3-hydroxy-1-oxobutan-2-yl]-19-[[(2r)-2-amino-3-naphthalen-2-ylpropanoyl]amino]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-7-propan-2-yl-1,2-dithia-5,8,11,14,17-p Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](N)CC=1C=C2C=CC=CC2=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)=O)C(C)C)C1=CC=C(O)C=C1 PUDHBTGHUJUUFI-SCTWWAJVSA-N 0.000 description 2
- ZWXONJFCJAGEBA-BXWFABGCSA-N 4-[2-(5-Hydroxy-2-methylphenyl)ethyl]-7a-methylhexahydro-1H-indene-1,5(4H)-dione Chemical compound CC1=CC=C(O)C=C1CC[C@@H]1C(=O)CC[C@]2(C)C(=O)CC[C@H]21 ZWXONJFCJAGEBA-BXWFABGCSA-N 0.000 description 2
- DFGKGUXTPFWHIX-UHFFFAOYSA-N 6-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]acetyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)C1=CC2=C(NC(O2)=O)C=C1 DFGKGUXTPFWHIX-UHFFFAOYSA-N 0.000 description 2
- 101710153593 Albumin A Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 102100020992 Interferon lambda-3 Human genes 0.000 description 2
- 101710099621 Interferon lambda-3 Proteins 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
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 101100042870 Rattus norvegicus Sst gene Proteins 0.000 description 2
- 101150084577 SST2 gene Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 102000021178 chitin binding proteins Human genes 0.000 description 2
- 108091011157 chitin binding proteins Proteins 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229910003460 diamond Inorganic materials 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 108010021336 lanreotide Proteins 0.000 description 2
- 229960002437 lanreotide Drugs 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 102000004052 somatostatin receptor 2 Human genes 0.000 description 2
- 108010049553 somatostatin receptor sst2A Proteins 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- NPJIOCBFOAHEDO-AVWFULIKSA-N (3s,6s,9s,12r,15s,18s)-9-(4-aminobutyl)-3-benzyl-15-[(4-hydroxyphenyl)methyl]-12-(1h-indol-3-ylmethyl)-1,18-dimethyl-6-propan-2-yl-1,4,7,10,13,16-hexazacyclooctadecane-2,5,8,11,14,17-hexone Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(N[C@@H](CC=2C=CC=CC=2)C(=O)N(C)[C@@H](C)C(=O)N1)=O)C(C)C)C1=CC=C(O)C=C1 NPJIOCBFOAHEDO-AVWFULIKSA-N 0.000 description 1
- SWXOGPJRIDTIRL-DOUNNPEJSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-n-[(2s)-1-amino-3-(1h-indol-3-yl)-1-oxopropan-2-yl]-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-7-propan-2-yl-1,2-dithia-5,8,11,14,17-pent Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](N)CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(N)=O)=O)C(C)C)C1=CC=C(O)C=C1 SWXOGPJRIDTIRL-DOUNNPEJSA-N 0.000 description 1
- KZEVSDGEBAJOTK-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[5-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CC=1OC(=NN=1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O KZEVSDGEBAJOTK-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical class OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- JQMFQLVAJGZSQS-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JQMFQLVAJGZSQS-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 108010091324 3C proteases Proteins 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- 208000003200 Adenoma Diseases 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000017701 Endocrine disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 102400000921 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 230000010799 Receptor Interactions Effects 0.000 description 1
- 108010076818 TEV protease Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000002016 colloidosmotic effect Effects 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000011523 endocrine gland cancer Diseases 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- -1 entrokinase Proteins 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000003629 gastrointestinal hormone Substances 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000000960 hypophysis hormone Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 230000008991 intestinal motility Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006662 intracellular pathway Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000004025 pancreas hormone Substances 0.000 description 1
- 108700017947 pasireotide Proteins 0.000 description 1
- 229960005415 pasireotide Drugs 0.000 description 1
- VMZMNAABQBOLAK-DBILLSOUSA-N pasireotide Chemical compound C([C@H]1C(=O)N2C[C@@H](C[C@H]2C(=O)N[C@H](C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@H](C(N[C@@H](CC=2C=CC(OCC=3C=CC=CC=3)=CC=2)C(=O)N1)=O)CCCCN)C=1C=CC=CC=1)OC(=O)NCCN)C1=CC=CC=C1 VMZMNAABQBOLAK-DBILLSOUSA-N 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 108010052231 seglitide Proteins 0.000 description 1
- 229950002758 seglitide Drugs 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229940075620 somatostatin analogue Drugs 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012536 storage buffer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 108700029852 vapreotide Proteins 0.000 description 1
- 229960002730 vapreotide Drugs 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention is related to fusion proteins of human somatostatin (e.g., SST-14 or SST-28) and human serum albumin, comprising a region at least 85% homologous to human somatostatin and a region at least 85% homologous to human serum albumin or a region with a partial amino acid sequence of human serum albumin, wherein linker peptide sequences may be present between somatostatin and somatostatin moieties or somatostatin and albumin moieties. Also disclosed are constructs wherein the somatostatin moiety contains multiple tandem repeats of a somatostatin sequence. In selected embodiments, the orientation of the somatostatin and albumin moieties can be varied, and such sequences may impact the binding and efficacy of the disclosed fusion proteins. Also disclosed are methods of making and using the aforementioned constructs. The somatostatin-albumin fusion protein demonstrated enhanced stability when incubated in rat plasma in vitro and prolonged plasma half-life in vivo compared with free somatostatin.
Description
COMPOSITIONS CONTAINING FUSION PROTEIN OF ALBUMIN AND
ANALOGS THEREOF, METHODS FOR MAKING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Patent Application 15/249,346 filed August 26, 2016, the contents of which are incorporated herein by reference.
U.S. Patent Application 15/249,346 is a continuation-in-part of PCT/U52016/019950, which claims the benefit of priority from U.S. Provisional Patent Application Serial No. US
62/121,487 filed on February 26, 2015, the contents of each of which are incorporated herein by reference.
U.S. Patent Application 15/249,346 also claims benefit from Taiwanese Patent Application No. 105106088 filed February 26, 2016, which also claims benefit of priority from U.S. Provisional Patent Application No. 62/121,487 filed on Feb 26, 2015, the contents of each of which are incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a fusion protein comprising a somatostatin, or its analogue or derivatives, a linker or spacer and an albumin, or its analogue or variant.
The present invention also relates to recombinant fusion proteins containing a human .. serum albumin moiety, and a somatostatin moiety, separated by a spacer sequence and analogues thereof.
BACKGROUND OF THE INVENTION
Somatostatin ("SST") is a secretory product of a variety of endocrine and non-.. endocrine tissues and is widely distributed throughout the body.
Somatostatin inhibits pituitary, pancreatic, and gastrointestinal hormone secretion release, as well as cytokine production, intestinal motility and absorption, vascular contractility, and cell proliferation.
Recent studies have found that SST has use as a treatment for cancer, inhibiting tumor growth, inhibiting the proliferation of endocrine tumors, and many other solid tumors, such .. as breast cancer, colorectal cancer, liver cancer, lung cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer and prostate cancer. The somatostatin molecule has two biologically active forms: somatostatin-14 (SST-14), the cyclic tetradecapeptide, and somatostatin-28 (SST-28), an N-terminally elongated form of SST-14. SST-14 is a cyclic peptide with a length of 14 residues, containing a disulfide linkage between cysteines at positions 3 and 14. SST-28 is an N-terminal extension form (28 residues) of the same precursor that is proteolytically cleaved to generate SST-14. Although the two have similar activity, their respective potency and histological characteristics vary. For example, SST-14 displays more pronounced inhibition of glucagon and gastrin, while SST-28 displays more pronounced inhibition of growth hormone and insulin action. Both forms of somatostatin exert their respective biological functions through receptors on target cells and intracellular pathways. Five subtypes of somatostatin receptors (SSTR 1-5) have been recognized, with two spliced variants for SSTR2: SSTR2A and SSTR2B, with a different carboxyl terminus.
The beneficial effects of somatostatin in the treatment of certain hypersecretory endocrine disorders, and its anti-proliferation effect on tumors are well recognized. However, the half-life of somatostatin in vivo is only 2-3 minutes due to enzymatic degradation and endocytosis, limiting clinical utility of somatostatin. In the past decade, numerous stable somatostatin analogs have been developed. For example, octreotide and lanreotide are used in treatment of growth hormone (GH)-secreting adenomas and carcinoids.
However, therapeutic limitations still exist due to altered binding affinity to SSTRs.
As a result, there remains a need in the art for somatostatin constructs that achieve high in vivo half-life while maintaining a desirable binding affinity to SSTRs.
Albumin, the most abundant protein in the blood plasma, is produced in the liver as a monomeric protein of 67 kDa and responsible for 80% of the colloid osmotic pressure of plasma. Human granulocyte colony stimulating factor (G- CSF), human growth hormone (GH), human insulin, human interferon-a-2b (INF-2b), and interleukin-28B (IL-28B) fused with HSA were used effectively to construct long-acting therapeutic drug candidates.
However, the comparative studies between HSA fusion proteins and the parent molecules in the biological and molecular mechanisms are less reported.
Chinese patent applications CN102391376A and CN102675467A, both hereby incorporated by reference, disclose somatostatin-albumin fusion proteins.
However, there remains a need for further development of somatostatin-albumin fusion proteins.
ANALOGS THEREOF, METHODS FOR MAKING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. Patent Application 15/249,346 filed August 26, 2016, the contents of which are incorporated herein by reference.
U.S. Patent Application 15/249,346 is a continuation-in-part of PCT/U52016/019950, which claims the benefit of priority from U.S. Provisional Patent Application Serial No. US
62/121,487 filed on February 26, 2015, the contents of each of which are incorporated herein by reference.
U.S. Patent Application 15/249,346 also claims benefit from Taiwanese Patent Application No. 105106088 filed February 26, 2016, which also claims benefit of priority from U.S. Provisional Patent Application No. 62/121,487 filed on Feb 26, 2015, the contents of each of which are incorporated herein by reference.
FIELD OF INVENTION
The present invention relates to a fusion protein comprising a somatostatin, or its analogue or derivatives, a linker or spacer and an albumin, or its analogue or variant.
The present invention also relates to recombinant fusion proteins containing a human .. serum albumin moiety, and a somatostatin moiety, separated by a spacer sequence and analogues thereof.
BACKGROUND OF THE INVENTION
Somatostatin ("SST") is a secretory product of a variety of endocrine and non-.. endocrine tissues and is widely distributed throughout the body.
Somatostatin inhibits pituitary, pancreatic, and gastrointestinal hormone secretion release, as well as cytokine production, intestinal motility and absorption, vascular contractility, and cell proliferation.
Recent studies have found that SST has use as a treatment for cancer, inhibiting tumor growth, inhibiting the proliferation of endocrine tumors, and many other solid tumors, such .. as breast cancer, colorectal cancer, liver cancer, lung cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer and prostate cancer. The somatostatin molecule has two biologically active forms: somatostatin-14 (SST-14), the cyclic tetradecapeptide, and somatostatin-28 (SST-28), an N-terminally elongated form of SST-14. SST-14 is a cyclic peptide with a length of 14 residues, containing a disulfide linkage between cysteines at positions 3 and 14. SST-28 is an N-terminal extension form (28 residues) of the same precursor that is proteolytically cleaved to generate SST-14. Although the two have similar activity, their respective potency and histological characteristics vary. For example, SST-14 displays more pronounced inhibition of glucagon and gastrin, while SST-28 displays more pronounced inhibition of growth hormone and insulin action. Both forms of somatostatin exert their respective biological functions through receptors on target cells and intracellular pathways. Five subtypes of somatostatin receptors (SSTR 1-5) have been recognized, with two spliced variants for SSTR2: SSTR2A and SSTR2B, with a different carboxyl terminus.
The beneficial effects of somatostatin in the treatment of certain hypersecretory endocrine disorders, and its anti-proliferation effect on tumors are well recognized. However, the half-life of somatostatin in vivo is only 2-3 minutes due to enzymatic degradation and endocytosis, limiting clinical utility of somatostatin. In the past decade, numerous stable somatostatin analogs have been developed. For example, octreotide and lanreotide are used in treatment of growth hormone (GH)-secreting adenomas and carcinoids.
However, therapeutic limitations still exist due to altered binding affinity to SSTRs.
As a result, there remains a need in the art for somatostatin constructs that achieve high in vivo half-life while maintaining a desirable binding affinity to SSTRs.
Albumin, the most abundant protein in the blood plasma, is produced in the liver as a monomeric protein of 67 kDa and responsible for 80% of the colloid osmotic pressure of plasma. Human granulocyte colony stimulating factor (G- CSF), human growth hormone (GH), human insulin, human interferon-a-2b (INF-2b), and interleukin-28B (IL-28B) fused with HSA were used effectively to construct long-acting therapeutic drug candidates.
However, the comparative studies between HSA fusion proteins and the parent molecules in the biological and molecular mechanisms are less reported.
Chinese patent applications CN102391376A and CN102675467A, both hereby incorporated by reference, disclose somatostatin-albumin fusion proteins.
However, there remains a need for further development of somatostatin-albumin fusion proteins.
2
3 PCT/US2017/039477 SUMMARY OF THE INVENTION
The present invention provides somatostatin-albumin fusion proteins and analogues thereof and methods of producing and using the same. Constructs prepared according to the invention include an albumin (or an analog thereof) moiety, a somatostatin moiety (SST-14, SST-28), and a spacer, such as a spacer or linker peptide, separating the two moieties.
A fusion protein according to the invention is also described as a polypeptide herein.
The polypeptide according to the invention may optionally include, in certain embodiments, one or more non-naturally occurring amino acids or amino acid residues.
The somatostatin-albumin fusion proteins and analogues thereof broadly include a human SST peptide moiety, a linker or spacer, and a human albumen moiety. The SST
peptide moiety can include analogues and derivatives thereofõ that actively inhibit the activity of human growth hormone. Optionally, the SST peptide moiety is obtained from natural or synthetic sources. The albumin moiety is, e.g., human albumin and/or active fragments or subdomains thereof. The linker or spacer is selected to enhance the stability of the somatostatin-albumin fusion protein. More particularly, the somatostatin-albumin fusion proteins and analogues thereof have a structure as follows.
The invention provides for a fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin, its analogue or derivative;
L is a spacer or a linker; and ALB is an albumin, its analogue or variant.
Preferably, the inventive fusion protein is isolated and purified.
Optionally, the ALB component of the inventive fusion protein is mammalian serum albumin. In one embodiment, the mammalian serum albumin is SEQ ID NO: 25, or a sequence having at least 85 % sequence identity thereto.
In other particular embodiments, the inventive fusion protein is selected from the group consisting of:
SST-(L)xi-ALB (I);
ALB -(L)x) -SST (II);
[SST-(L)x)] yi -ALB (III);
ALB- [(L)x) -SST] yl (IV);
[ S S T- (L)x 1] yi -ALB - [(L)x2-SST] y2 (V);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S ST] y3 (VII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB - [(L)x3-S ST] y2-(L)x) -ALB (IX);
and ALB-(L)xi-[SST-(L)x2]yi-ALB-RL)x3-SST]y2-(L)xi-ALB-RL)x4-S ST] y3 (X);
wherein, x 1, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, or more particularly from 1-5, or an integer from 1-4, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
In alternative embodiments, the inventive fusion protein comprises an SST that is either naturally occurring or synthetically manufactured.
In a further embodiment, the SST of the inventive fusion protein comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID
NOS: 17 or 18, respectively, or a sequence having at least 85% identity to either of these sequences.
The SST moiety is optionally SST-14 or SST-28.
In a further embodiment, the fusion protein comprises an L that is either flexible or alpha helically structured polypeptide linker or spacer.
In a further embodiment, the fusion protein comprises an L that is a polypeptide having 2-100 amino acids. The linkers or spacers according to a further embodiment of the invention encompass peptides covalently linked to somatostatin on one terminal and to albumin on another terminal.
The terms "linker" or "spacer" are used interchangeably herein to refer to short amino acid sequences used to separate multiple domains in a single protein. Absence of linkers between two or more discrete domains in a protein may result in reduced or improper functionality of the protein domains e.g., a reduction in catalytic activity or binding affinity
The present invention provides somatostatin-albumin fusion proteins and analogues thereof and methods of producing and using the same. Constructs prepared according to the invention include an albumin (or an analog thereof) moiety, a somatostatin moiety (SST-14, SST-28), and a spacer, such as a spacer or linker peptide, separating the two moieties.
A fusion protein according to the invention is also described as a polypeptide herein.
The polypeptide according to the invention may optionally include, in certain embodiments, one or more non-naturally occurring amino acids or amino acid residues.
The somatostatin-albumin fusion proteins and analogues thereof broadly include a human SST peptide moiety, a linker or spacer, and a human albumen moiety. The SST
peptide moiety can include analogues and derivatives thereofõ that actively inhibit the activity of human growth hormone. Optionally, the SST peptide moiety is obtained from natural or synthetic sources. The albumin moiety is, e.g., human albumin and/or active fragments or subdomains thereof. The linker or spacer is selected to enhance the stability of the somatostatin-albumin fusion protein. More particularly, the somatostatin-albumin fusion proteins and analogues thereof have a structure as follows.
The invention provides for a fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin, its analogue or derivative;
L is a spacer or a linker; and ALB is an albumin, its analogue or variant.
Preferably, the inventive fusion protein is isolated and purified.
Optionally, the ALB component of the inventive fusion protein is mammalian serum albumin. In one embodiment, the mammalian serum albumin is SEQ ID NO: 25, or a sequence having at least 85 % sequence identity thereto.
In other particular embodiments, the inventive fusion protein is selected from the group consisting of:
SST-(L)xi-ALB (I);
ALB -(L)x) -SST (II);
[SST-(L)x)] yi -ALB (III);
ALB- [(L)x) -SST] yl (IV);
[ S S T- (L)x 1] yi -ALB - [(L)x2-SST] y2 (V);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S ST] y3 (VII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S ST-(L)x2] yi -ALB - [(L)x3-S ST] y2-(L)x) -ALB (IX);
and ALB-(L)xi-[SST-(L)x2]yi-ALB-RL)x3-SST]y2-(L)xi-ALB-RL)x4-S ST] y3 (X);
wherein, x 1, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, or more particularly from 1-5, or an integer from 1-4, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
In alternative embodiments, the inventive fusion protein comprises an SST that is either naturally occurring or synthetically manufactured.
In a further embodiment, the SST of the inventive fusion protein comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID
NOS: 17 or 18, respectively, or a sequence having at least 85% identity to either of these sequences.
The SST moiety is optionally SST-14 or SST-28.
In a further embodiment, the fusion protein comprises an L that is either flexible or alpha helically structured polypeptide linker or spacer.
In a further embodiment, the fusion protein comprises an L that is a polypeptide having 2-100 amino acids. The linkers or spacers according to a further embodiment of the invention encompass peptides covalently linked to somatostatin on one terminal and to albumin on another terminal.
The terms "linker" or "spacer" are used interchangeably herein to refer to short amino acid sequences used to separate multiple domains in a single protein. Absence of linkers between two or more discrete domains in a protein may result in reduced or improper functionality of the protein domains e.g., a reduction in catalytic activity or binding affinity
4 for a receptor/ligand, due to the steric hindrance. Linking protein domains in the chimeric proteins using an artificial linker can increase the space between the domains. Preferably, the linker or spacer is selected independently of the somatostatin and albumin.
The linker L is either a flexible or alpha helically structured polypeptide linker or spacer. In certain embodiments, L contains at least one GGGGS, A(EAAAK)4A, (AP)n, wherein n is an integer selected from 10-34, (G)8, (G)5, or any combination thereof.
The albumin-somatostatin fusion constructs described herein may also include a signal peptide sequence ("SP"). Signal peptides are understood to refer to short amino acid sequences present at the N-terminus of a polypeptide that direct the cellular placement of a newly-synthesized protein. For example, signal peptides may lead to a protein being localized to a given intracellular region (e.g., the nucleus), inserted into a membrane (e.g., the cell membrane or the endoplasmic reticulum) or secreted from the cell. Besides directing localization, signal peptides may also be incorporated into recombinant proteins in order to improve stability, modify expression levels, and to aid in the proper folding of the recombinant proteins. The signal peptide sequence of the precursor protein is usually removed by signal peptidase in the host cell to produce a mature protein.
The albumin-somatostatin fusion constructs described herein may also include an affinity or purification tag as part of the polypeptide sequence to facilitate purification. Such tags are used as part of affinity chromatographic methods, e.g., high performance liquid chromatography (HPLC) in order to purify a protein sample from a crude biological source.
Suitable purification tags include, but are not limited to: poly-histidine (e.g., His-6 or H6), glutathione-S-transferase (GST), maltose-binding protein (MBP), chitin binding protein (CBP), FLAG-tag (FLAG octapeptide). When it is necessary to remove the affinity tag from the fusion protein, specific enzymatic cleavage site can be introduced in the linker region.
Enzymes commonly used for removal of affinity tags include, but are not limited to: factor Xa, entrokinase, thrombin, TEV protease, and rhinovirus 3C protease.
In a further embodiment, the invention provides a nucleotide sequence encoding a polypeptide comprising:
an SST;
an L; and an ALB,
The linker L is either a flexible or alpha helically structured polypeptide linker or spacer. In certain embodiments, L contains at least one GGGGS, A(EAAAK)4A, (AP)n, wherein n is an integer selected from 10-34, (G)8, (G)5, or any combination thereof.
The albumin-somatostatin fusion constructs described herein may also include a signal peptide sequence ("SP"). Signal peptides are understood to refer to short amino acid sequences present at the N-terminus of a polypeptide that direct the cellular placement of a newly-synthesized protein. For example, signal peptides may lead to a protein being localized to a given intracellular region (e.g., the nucleus), inserted into a membrane (e.g., the cell membrane or the endoplasmic reticulum) or secreted from the cell. Besides directing localization, signal peptides may also be incorporated into recombinant proteins in order to improve stability, modify expression levels, and to aid in the proper folding of the recombinant proteins. The signal peptide sequence of the precursor protein is usually removed by signal peptidase in the host cell to produce a mature protein.
The albumin-somatostatin fusion constructs described herein may also include an affinity or purification tag as part of the polypeptide sequence to facilitate purification. Such tags are used as part of affinity chromatographic methods, e.g., high performance liquid chromatography (HPLC) in order to purify a protein sample from a crude biological source.
Suitable purification tags include, but are not limited to: poly-histidine (e.g., His-6 or H6), glutathione-S-transferase (GST), maltose-binding protein (MBP), chitin binding protein (CBP), FLAG-tag (FLAG octapeptide). When it is necessary to remove the affinity tag from the fusion protein, specific enzymatic cleavage site can be introduced in the linker region.
Enzymes commonly used for removal of affinity tags include, but are not limited to: factor Xa, entrokinase, thrombin, TEV protease, and rhinovirus 3C protease.
In a further embodiment, the invention provides a nucleotide sequence encoding a polypeptide comprising:
an SST;
an L; and an ALB,
5 wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker; and ALB is an albumin or its analogues or variants.
In particular embodiments the inventive nucleotide encodes a polypeptide that is selected from the group consisting of, SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[S ST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX);
and ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein, each of xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, or more particularly from 1-5 or from 1-4, provided that there is at least one L present in the polypeptide.
In a further embodiment, the nucleotide sequence encodes a fusion protein wherein the SST comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or a sequence having at least 85%
identity to either of these sequences.
The invention is further contemplated to include an expression vector, e.g., a plasmid construct, a host cell comprising the expression vector, that is capable of expressing the inventive albumin-somatostatin fusion protein. The host cell can be a suitable bacterial host cell, a suitable mammalian host cell, a suitable plant host cell, or a suitable insect host cell.
The invention also provides for methods of treating a disease or disorder of endocrine release in a mammal, such as in a human subject, by administering an effective amount of a
L is a spacer or a linker; and ALB is an albumin or its analogues or variants.
In particular embodiments the inventive nucleotide encodes a polypeptide that is selected from the group consisting of, SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[S ST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)Ayi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX);
and ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein, each of xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, or more particularly from 1-5 or from 1-4, provided that there is at least one L present in the polypeptide.
In a further embodiment, the nucleotide sequence encodes a fusion protein wherein the SST comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or a sequence having at least 85%
identity to either of these sequences.
The invention is further contemplated to include an expression vector, e.g., a plasmid construct, a host cell comprising the expression vector, that is capable of expressing the inventive albumin-somatostatin fusion protein. The host cell can be a suitable bacterial host cell, a suitable mammalian host cell, a suitable plant host cell, or a suitable insect host cell.
The invention also provides for methods of treating a disease or disorder of endocrine release in a mammal, such as in a human subject, by administering an effective amount of a
6 pharmaceutical composition comprising the inventive fusion protein, wherein the disease or disorder of endocrine release is a condition that responds to the administration of somatostatin.
For example, the disease or disorder is a cancer selected from the group consisting of breast cancer, colorectal cancer, liver cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer, prostate cancer, brain cancer, and lung cancer. In certain embodiments, the cancer expresses somatostatin receptor type 1, 2, 3, 4 or 5.
It should also be understood that singular forms such as "a," "an," and "the"
are used throughout this application for convenience, however, except where context or an explicit statement indicates otherwise, the singular forms are intended to include the plural. Further, it should be understood that every journal article, patent, patent application, publication, and the like that is mentioned herein is hereby incorporated by reference in its entirety and for all purposes.
All numerical ranges should be understood to include each and every numerical point within the numerical range, and should be interpreted as reciting each and every numerical point individually. The endpoints of all ranges directed to the same component or property are inclusive, and intended to be independently combinable.
As used herein, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.
The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
The SST and albumin fusion proteins of present application provide advantages over natural SST of (a) higher in vivo stability, (b) higher binding affinity to SST receptors, (c) higher protein expression yield, and (d) better pharmacokinetic/pharmacodynamics behavior.
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
Unless defined
For example, the disease or disorder is a cancer selected from the group consisting of breast cancer, colorectal cancer, liver cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer, prostate cancer, brain cancer, and lung cancer. In certain embodiments, the cancer expresses somatostatin receptor type 1, 2, 3, 4 or 5.
It should also be understood that singular forms such as "a," "an," and "the"
are used throughout this application for convenience, however, except where context or an explicit statement indicates otherwise, the singular forms are intended to include the plural. Further, it should be understood that every journal article, patent, patent application, publication, and the like that is mentioned herein is hereby incorporated by reference in its entirety and for all purposes.
All numerical ranges should be understood to include each and every numerical point within the numerical range, and should be interpreted as reciting each and every numerical point individually. The endpoints of all ranges directed to the same component or property are inclusive, and intended to be independently combinable.
As used herein, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.
The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
The SST and albumin fusion proteins of present application provide advantages over natural SST of (a) higher in vivo stability, (b) higher binding affinity to SST receptors, (c) higher protein expression yield, and (d) better pharmacokinetic/pharmacodynamics behavior.
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.
Unless defined
7 otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the pharmacokinetic profile of SST in rat:
wherein the diamond (*) labeled curve represents the data from Rat #6;
the triangle (A) labeled curve represents the data from Rat #7;
the square (M) labeled curve represents the data from Rat #8;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured plasma concentration of SST at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST; and the "X" axis is plasma sampling time ('t") in hours.
Note that at certain time points, the detection of SST plasma concentration was below limits of quantitation FIG. 2 illustrates bi-phasic pharmacokinetic profile of SST Fusion Protein in rat (Black dotted line distinguishes between a-Phase (0-0.5 hour) and 13-Phase (0.75-4 hours):
wherein, the diamond (*) labeled curve represents the data from Rat #1;
the triangle (A) labeled curve represents the data from Rat #2;
the star (*) labeled curve represents the data from Rat #3;
the square (M) labeled curve represents the data from Rat #4;
the "x" ( x ) labeled curve represents the data from Rat #5;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured plasma concentration of SST fusion protein at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST
fusion protein; and the "X" axis is plasma sampling time ('t") in hours.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the pharmacokinetic profile of SST in rat:
wherein the diamond (*) labeled curve represents the data from Rat #6;
the triangle (A) labeled curve represents the data from Rat #7;
the square (M) labeled curve represents the data from Rat #8;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured plasma concentration of SST at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST; and the "X" axis is plasma sampling time ('t") in hours.
Note that at certain time points, the detection of SST plasma concentration was below limits of quantitation FIG. 2 illustrates bi-phasic pharmacokinetic profile of SST Fusion Protein in rat (Black dotted line distinguishes between a-Phase (0-0.5 hour) and 13-Phase (0.75-4 hours):
wherein, the diamond (*) labeled curve represents the data from Rat #1;
the triangle (A) labeled curve represents the data from Rat #2;
the star (*) labeled curve represents the data from Rat #3;
the square (M) labeled curve represents the data from Rat #4;
the "x" ( x ) labeled curve represents the data from Rat #5;
the "Y" axis is Ln(Ct/C0) that represents the natural log of the measured plasma concentration of SST fusion protein at time;
"t" (Ct) divided by the initial measured plasma concentration (CO) of SST
fusion protein; and the "X" axis is plasma sampling time ('t") in hours.
8 DETAILED DESCRIPTION OF THE INVENTION
The present invention encompasses somatostatin-albumin fusion proteins and analogues thereof and methods of producing and using the same. Constructs prepared according to the invention include an albumin (or an analog thereof) moiety, a somatostatin moiety (e.g., SST-14, SST-28), and a spacer separating the two moieties.
The somatostatin-albumin fusion proteins of the certain embodiment of the invention include variants of albumin including human serum albumin and / or derivatives of somatostatin. The spacers of another embodiment of the invention encompass peptides covalently linked to somatostatin on one terminal and albumin on another terminal. The spacers in other embodiments of the invention include peptide sequences having 2-100 amino acids.
In one embodiment, the present invention provides a fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the fusion protein of the present invention is selected from among formulas I-X, as follows.
SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[ S ST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX); and
The present invention encompasses somatostatin-albumin fusion proteins and analogues thereof and methods of producing and using the same. Constructs prepared according to the invention include an albumin (or an analog thereof) moiety, a somatostatin moiety (e.g., SST-14, SST-28), and a spacer separating the two moieties.
The somatostatin-albumin fusion proteins of the certain embodiment of the invention include variants of albumin including human serum albumin and / or derivatives of somatostatin. The spacers of another embodiment of the invention encompass peptides covalently linked to somatostatin on one terminal and albumin on another terminal. The spacers in other embodiments of the invention include peptide sequences having 2-100 amino acids.
In one embodiment, the present invention provides a fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the fusion protein of the present invention is selected from among formulas I-X, as follows.
SST-(L)xi-ALB (I);
ALB-(L)xi-SST (II);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi]yi-ALB-[(L)x2-SST] y2 (V);
[ S ST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB (VI);
[SST-(L)xi]yi-ALB-[(L)x2-SST]y2-(L)o-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)xi-[SST-(L)x2]yi-ALB (VIII);
ALB-(L)xi- [SST-(L)x2]yi-ALB-[(L)3-SST]y2-(L)xi-ALB (IX); and
9 ALB-(L)) - [ S S T-(L)x2] yi -ALB - [(L)x3-SS T] y2-(L)x 1 -ALB - [(L)x4-S S
T] y3 (X);
wherein, each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the fusion protein.
In yet another embodiment, the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the nucleotide sequence of the present invention is selected to encode an albumin-somatostatin fusion protein from among, SST-(L)xi-ALB (I);
ALB-(L)xi-SST (11);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi ] yi -ALB - [(L)x2.-SS 1] y2 (V);
[SST-(L)x)] yi -ALB - [(L)x2.-SS 1] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S S T] y3 (VII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB - [(L)x3-S S 1] y2-(L)x) -ALB (IX); and ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein, each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, 1-5 or 1-4, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
Another embodiment of the present invention provides a nucleotide sequence .. encoding an albumin-somatostatin fusion protein, wherein the spacer sequence consists of the sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or ¨GGGGS-.
Another certain embodiment of the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein, wherein the second region (b) encodes a polypeptide having at least 85% sequence identity to SEQ ID NO: 19, albumin or a fragment .. thereof.
One embodiment of the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein, wherein the first region (a) encodes a polypeptide having at least 85% sequence identity to either SEQ ID NOS: 17 or 18, SST-14, SST-28, or a fragment thereof.
The present invention also provides a nucleotide sequence encoding an albumin-somatostatin fusion protein comprising:
(a) a first region comprising a nucleotide sequence containing one or more adjacent repeats of a sequence encoding a human somatostatin peptide;
(b) a second region comprising a nucleotide sequence encoding human serum albumin, or a fragment thereof;
(c) a spacer region comprising a nucleotide sequence encoding a polypeptide of residues in length;
wherein the spacer region is present between the first region and the second region, or or between the first region and another first region;
wherein one or more adjacent repeats of a sequence encoding a human somatostatin peptide encodes either SST-14 or SST-28, as represented by SEQ ID NOS:17 and 18, respectively, or a sequence having at least 85% identity to either of these two sequences; or wherein the spacer sequence consists of the sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or GGGGS or by SEQ ID NO: 30 A(EAAAK)4A;
or wherein the region (a) consists of one or more adjacent repeats of either SST-14 or of SST-28, as represented by SEQ ID NOS: 23 and 24, respectively, or a sequence having at least 85% identity to either of these two sequences.
Furthermore, the present invention provides a polypeptide sequence an albumin-somatostatin fusion protein comprising:
(a) a first region comprising a polypeptide sequence of a somatostatin peptide (which may be a human somatostatin peptide);
(b) a second region comprising a polypeptide sequence of serum albumin (which may be a human serum albumin), or a fragment thereof;
(c) a spacer region comprising a polypeptide of 2-100 residues in length.
The spacer region (c) may be present between region (a) and region (b) or between region (a) and region (a). In addition, the region (a) may comprise one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or sequence having 85% identity to either of these sequences.
Another embodiment of the present invention provides a plasmid construct expressing an albumin-somatostatin fusion protein with any of the fusion protein or polypeptide sequences described above.
Yet another embodiment of the present invention includes a bacterial cell transformed with the plasmid construct described above.
A further embodiment of the present invention includes an isolated and purified albumin-somatostatin fusion protein having the polypeptide sequence described above (e.g., a polypeptide sequence of an albumin-somatostatin fusion protein or the plasmid construct expressing such protein).
Table 1. A non-exclusive list of polypeptide sequences SEQ ID NO: Description SEQ ID NO: 1 55T14-A(EAAAK)4A-HSA-A(EAAAK)4A-55T14 SEQ ID NO: 2 HSA-A(EAAAK)4A-55T14 SEQ ID NO: 3 His6-GGS-HSA-GGGGS-55T14-HSA
SEQ ID NO: 4 His6-GGS-HSA-GGGGS-(55T14-GGGGS)2-HSA
SEQ ID NO: 5 HSA-GGGGS-(55T14-GGGGS)2- HSA
SEQ ID NO: 6 Linker GGGGGGGG
SEQ lD NO: 7 SST14-(GGGGS)3-HSA
SEQ lD NO: 8 SST14-A(EAAAK)4A-HSA
SEQ lD NO: 9 His6-GGS-HSA-GGGGS-55T14 SEQ lD NO: 10 55T14-GGGGS-HSA-GGS-His6 SEQ lD NO: 11 HSA-GGGGS-55T14 SEQ lD NO: 12 55T14-GGGGS-HSA
SEQ lD NO: 13 (55T14-GGGGS)2- HSA
SEQ lD NO: 14 (55T14-GGGGS)4- HSA
SEQ lD NO: 15 HSA-(GGGGS)3-55T14 SEQ lD NO: 16 HSA-(GGGGS)6-55T14 SEQ ID NO: 17 Somatostatin-14 (SST-14) SEQ ID NO: 18 Somatostatin-28 (SST-28) SEQ ID NO: 19 Human Serum Albumin (HSA) SEQ lD NO: 20 MDMRVPAQLLGLLLLWLRGARC (Signal Peptide) SEQ lD NO: 21 Linker APAPAPAPAPAPAPAPAPAP
SEQ lD NO: 22 Linker APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
SEQ lD NO: 30 A(EAAAK)4A peptide SEQ lD NO: 31 GGGGS peptide SEQ ID NO: 32 Linker GGGGSLVPRGSGGGGS
SEQ lD NO: 33 Linker GSGSGS
SEQ lD NO: 34 Linker GGGGSLVPRGSGGGG
SEQ ID NO: 35 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 36 Linker GGSGGHMGSGG
SEQ ID NO: 37 Linker GGSGGSGGSGG
SEQ ID NO: 38 Linker GGSGGHMGSGG
SEQ ID NO: 39 Linker GGSGG
SEQ ID NO: 40 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 41 Linker GGSGGGGG
SEQ ID NO: 42 Linker GSGSGSGS
SEQ ID NO: 43 Linker GGGSEGGGSEGGGSEGGG
SEQ ID NO: 44 Linker AAGAATAA
SEQ ID NO: 45 Linker GGGGG
SEQ ID NO: 46 Linker GGSSG
SEQ ID NO: 47 Linker GSGGGTGGGSG
SEQ ID NO: 48 Linker GSGSGSGSGGSGGSGGSGGSGGSGGS
For the fusion proteins, e.g., SEQ ID NOs: 1-5, 7-10 and 13-16, it should be noted that these are encoded as pro-proteins with a 22 residue signal peptide (SEQ ID NO: 20).
Somatostatin-Albumin Fusion Proteins The invention encompasses polypeptide constructs wherein the somatostatin moiety is encoded by a nucleotide having at least 85% sequence identity to the nucleotide sequence of endogenous human SST-14 or SST-28 (SEQ ID Nos: 23 and 24, respectively).
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety is encoded by a nucleotide having at least 85% sequence identity to the nucleotide sequence of endogenous human serum albumin (SEQ ID NO: 25). The invention further encompasses polypeptide constructs wherein the human serum albumin moiety is a fragment of the endogenous human serum albumin protein, e.g., where it is encoded by a nucleotide consisting of a subsequence of SEQ ID NO: 25. For example, the human serum albumin fragment optionally includes one or more of the three human serum albumin globular domains, each of which contains two subdomains, denominated subdomain IA, TB, IIA, JIB, IIIA, and IIIB (Dockal, 1999, The Journal Of Biological Chemistry, 274(41):
29303-29310).
The invention also encompasses polypeptide constructs wherein the somatostatin moiety has a polypeptide sequence at least 85% sequence identity, preferably at least 90% to the polypeptide sequence of endogenous SST-14 or SST-28 (SEQ ID NOs:17 and 18, respectively).
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety has a polypeptide sequence at least 85% sequence identity to the polypeptide sequence of mature human serum albumin (SEQ ID NO: 19).
The invention also encompasses a fusion protein comprising a signal peptide, a purification tag (His-6), a first linker, a human serum albumin moiety, a second linker and a somatostatin moiety. In one embodiment, the fusion protein is a polypeptide is represented by SEQ ID NO: 9 or a sequence having 85% sequence identity to the same.
The invention also encompasses a fusion protein comprising a somatostatin moiety, a first linker, a human serum albumin moiety, a second linker, a somatostatin moiety and a purification tag (His-6). In one embodiment, the fusion protein is a polypeptide is represented by SEQ ID NO: 10 or a sequence having 85% sequence identity to the same.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 11) encoding a fusion protein comprising an N-terminal human serum albumin moiety and a C-terminal somatostatin moiety separated by a peptide spacer. The invention further encompasses nucleotide sequences encoding an albumin-somatostatin fusion construct which have 85%
sequence identity to SEQ ID NO: 11.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 12) encoding a fusion protein comprising an N-terminal somatostatin moiety and a C-terminal human serum albumin moiety separated by a peptide spacer. The invention further encompasses nucleotide sequences encoding an albumin-somatostatin fusion construct which have 85%
sequence identity to SEQ ID NO: 12.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more copies of the SST-14 or SST-28 sequence arranged in tandem, i.e., "(SST-14)2" or "(SST-14)3"or "(SST-28)2" or "(SST-28)3", respectively.
Optionally, a linker sequence is included between the two or more tandem somatostatin moieties, and/or a signal peptide sequence is included at the N-terminus of the fusion protein.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more copies of the SST-14 sequence arranged in a way that at least one copy of the SST14 is linked on both sides of albumin, respectively.
Optionally, a linker sequence is included between the two or more tandem somatostatin moieties and between somatostatin and albumin, and/or a signal peptide sequence is included at the N-terminus of the fusion protein. For example, the polypeptide construct may include a signal peptide, two SST-14 moieties separated by a spacer, a second spacer, and an HSA moiety as represented.
Optionally, the construct omits the N-terminal signal peptide.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or three copies of the SST-28 sequence arranged in tandem, i.e., "(SST-28)2" or "(SST-28)3", respectively. Optionally, a linker sequence is included between the two or more tandem somatostatin moieties.
The invention also encompasses polypeptide constructs comprising any of the albumin-somatostatin fusion proteins described in the preceding paragraphs, where the albumin-somatostatin fusion protein has an in vivo half-life longer than the endogenous SST-14 or SST-28 peptides.
The invention also encompasses polypeptide constructs comprising any of the albumin-somatostatin fusion proteins described in the preceding paragraphs, wherein the albumin-somatostatin fusion protein has an approximately equal or a greater binding affinity for a somatostatin receptor compared to endogenous SST-14 or SST-28.
The invention also encompasses albumin-somatostatin fusion proteins comprising an N-terminal albumin moiety as represented by SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID
NO: 2, an internal SST moiety and a C-terminal Albumin moiety as represented by SEQ ID
NO: 7 and SEQ ID NO: 8. Optionally, the N-terminus may further include a signal peptide.
Optionally, one or more of the albumin and SST domains may each be separated by an independently selected linker sequence as represented by SEQ ID NO: 1.
In some embodiments, the SST moiety may comprise a pair or plurality of tandem SST sequences, e.g., (SST-14)2 or (SST-28)3, with or without intervening spacing sequences between the two or more tandem SST repeats. Optionally, one or more purification tag sequences may be included in the sequence between two moieties or at the N or C-terminus in order to assist with purification of the fusion protein. An alternative embodiment includes a pair of SST-14 moieties separated by a spacer, as represented by SEQ ID NO:
4. A further embodiment may omit the purification tag (e.g., His6) as shown by the polypeptide sequence represented by SEQ ID NO: 5.
Somatostatin The somatostatin for use with the present invention may be any somatostatin, its analogue or derivative. It may be a human somatostatin, any other isolated or naturally occurring somatostatin. The SST moiety can be an analogue such as octreotide, lanreotide, pasireotide, seglitide, or vapreotide.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises a somatostatin analog. Preferably, such an analog is suitable for expression, as part of a fusion protein, in a recombinant host cell. It is understood that a suitable somatostatin analog sequence may be used in place of the SST-14 or sequences included in any of the examples disclosed herein.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more tandem repeats of a somatostatin polypeptide sequence e.g., SST-14 or SST-28; SEQ ID NOS: 17 and 18, respectively. Each of the repeated somatostatin polypeptide sequences may be a polypeptide sequence having at least 85%
sequence identity to SST-14 or SST-28. These repeated variant sequences are independently selected, i.e., in some embodiments the repeats are identical, whereas in other embodiments they are unique.
Albumin The albumin for use with the present invention may be any albumin, its analogue or variant. The albumin may be human serum albumin, or any other isolated or naturally occurring albumin.
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety comprises a polypeptide sequence variant with alternative arrangement or number of disulfide bonds due to the presence of additional or fewer cysteine residues than the natural form (SEQ ID NO: 25).
Spacer or Linker As described earlier, a spacer or linker can be used with the present invention. The spacer or linker may be independent of the somatostatin or albumin.
The invention also encompasses polypeptide constructs wherein the peptide spacer of alternatively referred to as a linker, consists of a polypeptide sequence of from about 2 to about 100 amino acid residues in length. The invention further encompasses polypeptide constructs wherein the peptide spacer is from about 2 to about 50 amino acid residues in length, preferably from about 2 to about from 30, or more preferably from about 3 to about 20 amino acid residues in length.
The invention also encompasses polypeptide constructs wherein the peptide spacer (alternatively referred to as a linker) has the polypeptide sequence "GGGGS"
(SEQ ID NO:
31). Polypeptides rich in Gly, Ser or Thr offer special advantages include, but not limited to:
(i) rotational freedom of the polypeptide backbone, so that the adjacent domains are free to move; (ii) enhanced solubility; (iii) resistance to proteolysis. In addition, many natural linkers exhibited alpha-helical structures. The alpha-helical structure is more rigid and stable than Gly rich linker. An empirical rigid linker with the sequence of A(EAAAK)4A
(SEQ ID NO:
30) can be used to separate functional domains. In addition to the role of linking protein domains together, artificial linkers may offer other advantages to the production of fusion proteins, such as improving biological activity, increasing protein expression, and achieving desirable pharmacokinetic profiles.
Table 2. A non-exhaustive list of linker sequences that may be used in the fusion protein constructs of the present invention.
GGGGSLVPRGSGGGGS (SEQ ID NO: 32) GSGSGS (SEQ ID NO: 33) GGGGSLVPRGSGGGG (thrombin proteolytic site is underlined) _ (SEQ ID NO: 34) GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined) _ (SEQ ID NO: 35) GGSGGHMGSGG (SEQ ID NO: 36) GGSGGSGGSGG (SEQ ID NO: 37) GGSGGHMGSGG (SEQ ID NO: 38) GGSGG (SEQ ID NO: 39) GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined) (SEQ ID NO: 40) GGSGGGGG (SEQ ID NO: 41) GSGSGSGS (SEQ ID NO: 42) GGGSEGGGSEGGGSEGGG (SEQ ID NO: 43) AAGAATAA (SEQ ID NO: 44) GGGGG (SEQ ID NO: 45) GGSSG (SEQ ID NO: 46) GSGGGTGGGSG (SEQ ID NO: 47) GT
GSGSGSGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 48) GGS
GGGGGGGG (SEQ ID NO: 6) A(EAAAK)4A (SEQ ID NO: 20) APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 21) APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
(SEQ ID NO: 22) Preparation of Somatostatin-Albumin fusion protein An embodiment of the invention provides a method for preparation of the somatostatin-albumin fusion protein. In one embodiment, the somatostatin-albumin fusion protein of the invention is prepared by expressing a vector containing the encoding gene and introducing the vector into a suitable host cell. For example, the fusion protein is obtained by expression of a suitable vector in a host such as yeast. In one embodiment, Pichia pastoris GS115 may be used as a suitable expression host, and the vector is pPIC9K. In particular, mammalian cell lines such as CHO or HEK293 can be used as an expression host.
The invention also encompasses plasmid constructs capable of expressing an albumin somatostatin fusion protein comprising a nucleotide sequence encoding a somatostatin albumin fusion protein as described in any of the preceding paragraphs. For example, suitable plasmid constructs include, but are not limited to, the pcDNA3.1 vector represented by SEQ ID NO: 26 with a DNA sequence encoding any of the albumin-somatostatin fusion proteins disclosed herein ligated into the multiple cloning site of this vector. Other suitable protein expression vectors known in the art may be selected based upon the expression host (e.g., an expression vector with a mammalian promoter system would be suitable for expression in a human cell line whereas a yeast or bacterial expression plasmid would be selected if expression in either of these organisms was desired.
The invention also encompasses a bacterial or yeast protein expression system comprising a bacterial or yeast cell transformed with a plasmid construct comprising a nucleotide sequence that encodes a somatostatin albumin fusion protein, as described in any of the preceding paragraphs. Suitable bacterial strains include, for example, Escherichia coli.
Suitable yeast strains include, for example, Pichia pastoris. An exemplary plasmid construct .. includes pPIC9K (Invitrogen) as represented by SEQ ID NO: 27, with a nucleotide sequence encoding any of the albumin-somatostatin fusion proteins described herein incorporated into the multiple cloning site of the vector.
The invention also encompasses isolated and purified fusion somatostatin fusion proteins having a polypeptide sequences as described in any of the preceding paragraphs.
Table 3. A list of nucleotide sequences in certain embodiments of the invention Nucleotide Sequence Encodes the SEQ ID NO: Description following:
SEQ ID NO: 23 55T14 Somatostatin-14 (SST-14) SEQ ID NO: 24 55T28 Somatostatin-28 (SST-28) Human Serum Albumin SEQ ID NO: 25 Human Serum Albumin mature form (HSA) pcDNA3.1(+) Vector SEQ ID NO: 26 pcDNA3.1(+) Vector mammalian expression vector SEQ ID NO: 27 pPIC9K Vector yeast expression vector SEQ ID NO: 28 GGGGS GGGGS Linker SEQ ID NO: 29 A(EAAAK)4A alpha-helical linker When the SST is a somatostatin analogue, an alternative method known in the field can be employed to prepare the conjugate.
Utility of Somatostatin-Albumin Fusion Protein The fusion protein of the present invention can be used to treat conditions for which somatostatin is art-known to be employed. As such, the invention also encompasses methods of treating cancer in a human subject by administering an isolated and purified albumin-somatostatin fusion protein as described in any of the preceding paragraphs, wherein the cancer is any cancer known to respond to somatostatin treatment, e.g., selected from breast cancer, colorectal cancer, liver cancer, lung cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer and prostate cancer.
The invention also encompasses methods of treating cancer in a human subject by administering a composition containing the fusion protein of the present invention, such as an isolated and purified albumin-somatostatin fusion protein as described in any of the preceding paragraphs. The composition can also include a suitable carrier.
Eleven 55T14-Albumin fusion protein constructs with various linker sequences were designed. Eight of these constructs were made into a fusion gene within a plasmid and produced by HEK 293 transient expression at 100 mL scale. The proteins were collected from the culture media, purified through albumin-based affinity purification, and dialyzed to a storage buffer. These fusion proteins were evaluated for their binding affinity to SSTR2 receptor, and also for cell-based activity in inhibiting cAMP production in a overexpression CHO-Kl cell line. The results of these studies indicated that the length and type of linkers significantly affected the SSTR2 receptor binding affinity, the in-vitro cell-based functional activity, and the fusion protein production yield.
SST-Albumin fusion protein of this invention exhibited a significantly longer serum half-life and/or improved pharmacokinetic profile in solution or in a pharmaceutical composition in vitro and/or in vivo compared to the corresponding unfused, free SST
molecules. The stability of free SST and SST fusion protein was compared in in vitro rat plasma. When incubated in freshly prepared rat plasma at 37 C, free SST and SST fusion protein exhibited degradation half-lives of 33 minutes and 5.5 hours, respectively (Table 4).
In vivo pharmacokinetic profiles were also generated to demonstrate the improved stability of SST fusion protein relative to free SST. Rats administered intravenously with SST showed a calculated T1/2 of 3.5 minutes. On the other hand, rats administered intravenously with SST fusion protein exhibited a bi-phasic pharmacokinetic profile, where the a-phase Ti/2 was 1.01 hour and the 0-phase Ti/2 was 6.14 hour. The calculated half-life of SST fusion protein is significantly longer than the calculated T1/2 of free SST in rat (3.5 .. minutes) and the reported plasma Ti/2 of free SST in rat (< 1 minute;
Reference #1) (Table 4).
Table 4. Calculated Half-life of Free SST and SST Fusion Protein for In Vitro Plasma Stability and In Vivo Rat Model T1/2 In Vitro Plasma Stability In Vivo Rat Pharmacokinetic Free SST 33 minutes 3.5 1.0 minutes less than 1 minutes (Ref. #1) a phase (0-0.5 hr) 1.01 0.60 hr SST Fusion Protein 5.5 hour [3 phase (0.75 - 4 hr) 6.14 1.27 hr Reference #1: Yogesh C. Patel and Thomas Wheatley. In Vivo and in Vitro Plasma Disappearance and Metabolism of Somatostatin-28 and Somatostatin-14 in the Rat.
Endocrinology. Vol. 112, No. 1(1992), pages 220-225.
EXAMPLES
Selected embodiments of the invention will be described in further detail with reference to the following experimental and comparative examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLE 1: EXPRESSION IN MAMMALIAN SYSTEMS
Example 1-1. Recombinant gene synthesis Eight constructs corresponding to the fusion proteins listed in Table 5 were prepared.
First, the gene sequence coding each fusion protein was de novo synthesized and subsequently inserted into the pcDNA3.1 vector.
Example 1-2. Plasmid generation Maxi-prep or Mega-prep was used to generate ¨20 mg of each DNA
Example 1-3. Transfection and protein production (A) Suspension cell method FreeStyleTM 293-F Cells were seeded at 0.55-0.6x106 cells/mL in a flask. After about 24 hours, the cells were seeded in a shake flask at 1.1-1.2x106 cells/mL. DNA
was prepared at 500 g DNA / 80 mL in a FreeStyle medium. Polyethylenimine (PEI) was prepared at 1.8 mL PEI per 80 mL in a FreeStyle medium. DNA was mixed in the FreeStyle medium, and the effective amount of PEI was added to the DNA solution, and the mixture is vortexed incubated for about 15 minutes at room temperature to form a DNA-PEI complex.
An 80 mL
of the incubated DNA-PEI complex is added to a cell culture. About 3 hours later, TC
Yeastolate feed (BD) is added to have the final concentration of 4 gram /
liter of culture.
After about 7-8 days, the medium is harvested by centrifugation.
(B) Adherent cell method About 24 hours before transfection, HEK293 cells were seeded to 50-90 %
confluency in a flask, and complete medium is added. After about 24 hours, cells were washed followed by adding basal medium.
DNA and PEI solutions were prepared by adding DNA to a serum free medium. The PEI solution was added to the DNA solution and incubated for 15 minutes to form DNA-PEI
complex at room temperature.
The PEI/DNA mixture was added to cells, and the mixture incubated for about 4-hours at 37 C. The medium was removed and fresh medium with Glutamine and serum was added, followed by incubating at 37 C for 4 days.
The medium was harvested after about 4 days, by centrifuging to collect the supernatant. The precipitate was replenished with fresh medium with L-Glutamine for another 3-day incubation to repeat the harvesting process.
Example 1-4: Protein Concentration, Ni-NTA Purification and Buffer Exchange The collected medium was concentrated by TFF system (Millipore) to a certain volume depending on purification methods (either continuous chromatography or manual batch purification).
The concentrated proteins was incubated with fresh Ni-NTA resin at about 4 C
in binding buffer and washed with wash buffer using either chromatography or batch system.
The protein was eluted with elute buffer and fractions were collected and concentrated to recover the purified protein. The protein can be further purified using size exclusion chromatography purification.
The buffer of the final eluate can be exchanged by dialysis to a desired buffer.
EXAMPLE 2: YIELDS OF SEVERAL SST-ALBUMIN FUSION PROTEINS
The SST-HSA fusion proteins were all expressed in soluble form with high yield. The length or the nature of the linkers can affect the protein yield and solubility of the fusion proteins. The results indicated that the production yield slightly decreased as the fusion protein constructs became longer and more complex. However, all the constructs exhibited yield for scale up production.
Table 5. SST14-HSA fusion protein expression yield Total Production Sequence ID Design amino MW (kDa) Yield acids (g/L) SST14-A(EAAAK)4A-HSA-SEQ ID NO: 1 657 73.8364 0.26 A(EAAAK)4A-SST14 SEQ ID NO: 2 HSA A(EAAAK)4A-55T14 621 70.1543 0.27 SEQ ID NO: 7 55T14-(GGGGS)3-HSA 614 69.112 0.33 SEQ ID NO: 8 55T14-A(EAAAK)4A-HSA 621 70.1543 0.25 SEQ ID NO: 9 613 69.4874 0.30 SEQ ID NO: 10 613 69.4874 0.41 SEQ ID NO: 15 HSA-(GGGGS)3-55T14 614 69.112 0.28 SEQ ID NO: 16 HSA-(GGGGS)6-55T14 629 70.1119 0.29 EXAMPLE 3: BINDING AFFINITY OF SEVERAL SST-ALBUMIN FUSION
PROTEINS
This assay measures binding of [125I]Somatostatin to human somatostatin sst2 receptors. CHO-Ki cells stably transfected with a plasmid encoding the human somatostatin sst2 receptor are used to prepare membranes in modified HEPES pH 7.4 buffer using standard techniques. A 0.1 mg aliquot of membrane is incubated with 0.03 nM
[125I]Somatostatin and tested fusion proteins for 240 minutes at 25 C. Non-specific binding is estimated in the presence of 1 v.1\4 Somatostatin. Membranes are filtered and washed 3 times and the filters are counted to determine [125I]Somatostatin specifically bound.
The competitive binding study 125I-Tyr-somatostatin versus the fusion proteins demonstrated the following results. The efficiency of the inhibition varied depending on the construct of the fusion proteins. The fusion protein construct (SEQ ID NO: 1) with two alpha-helical linker, A(EAAAK)4A, showed 100 % inhibition of the somatostatin and its receptor interaction. The SEQ ID NO: 1 construct has two somatostatin moiety on both N
and C terminal sides of human serum albumin. The smaller construct with one somatostatin on the C terminal side of human serum albumin linked by the same alpha-helical linker (SEQ
ID NO: 2) showed 96 % inhibition. The same construct with the more flexible GGGGS
linker showed lower inhibition of 82 ¨ 85 % depending on the length. The length of GGGGS
linkers also affected the inhibition. The construct with five amino acid GGGGS
linker (SEQ
ID NO: 9 and SEQ ID NO: 8) showed 57-59 % inhibition whereas the constructs with 15 amino acid (SEQ ID NO: 15) or 30 amino acid GGGGS linkers (SEQ ID NO: 16) showed over 80 %, suggesting that longer than five amino acid GGGGS would be more advantageous to SST function. A more rigid A(EAAAK)4A (a-helical) linker would be more efficient in binding than flexible GGGGS linker. A multiple SST can increase the effective concentration of the ligand for SST receptor binding. The position of Histidine purification tag may not affect the binding. Changing the orientation or position of albumin in the fusion protein may further increase the efficiency of the protein binding.
Table 6. Inhibition of 125I-Tyr1-somatostatin binding on SSTR2 by the fusion proteins Inhibition %
Sequence ID Construct ICso (nM) at 0.1 p,M
SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 100 2.38 A(EAAAK)4A-55T14 SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 96 9.41 SEQ ID NO: 7 55T14-(GGGGS)3-Albumin 70 SEQ ID NO: 8 55T14-A(EAAAK)4A-Albumin 79 SEQ ID NO: 9 His6-GGS-Albumin-GGGGS-55T14 59 SEQ ID NO: 10 55T14-GGGGS-Albumin-His6 57 SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 85 33 SEQ ID NO: 16 Albumin-(GGGGS)6-55T14 82 SEQ ID NO: 17 SST-14 0.0069 EXAMPLE 4. INHIBITION OF SEVERAL SST-ALBUMIN FUSION PROTEINS TO
Human recombinant somatostatin sst2a receptors expressed in CHO-Kl cells were used. Test compound and/or vehicle was incubated with the cells (2 x 105 cells/mL) in incubation buffer for 20 minutes at 37 C. Test compound-induced decrease of cAMP by 50 percent or more (50%) relative to the 10 nM Octreotide response indicated sst2a receptor agonist activity.
The inhibition of the accumulation of cAMP was observed in SST receptor type 2 expressing CHO-Kl cells. The value of EC50 was 260 nM. The constructs with longer linkers (SEQ ID NOS: 1, 15, and 2) exhibited lower EC50 values, which coincided with the binding assay data. The alpha-helical linker appeared to be more efficient in the inhibition of cAMP
production, when Albumin-(GGGGS)3-SST14 and Albumin-(GGGGS)3-SST14 EC50 values were compared.
Table 7. EC50 value for the inhibition of cAMP production ECso values of the Sequence ID Construct inhibition of cAMP
production (nM) SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 5.14 A(EAAAK)4A-55T14 SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 17.6 SEQ ID NO: 9 H6-GGGGS-Albumin-GGGGS-55T14 260 SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 23 Octreotide 0.041 EXAMPLE 5. DETERMINATION OF STABILITY OF FREE SST AND SST
FUSION PROTEIN IN RAT PLASMA
Improved stability of SST fusion protein in rat plasma was proven using ELISA.
The test results showed that SST fusion protein (SEQ ID NO: 1) exhibited a degradation half-life of 5.5 hours as opposed to free SST, which showed less than 33 minute half-life when incubated in rat plasma at 37 C (Table 4).
Example 5-1: Preparation of Sample 500 pg/mL of free SST or with 750 ng/mL of SST fusion protein (SEQ ID NO: 1) were incubated in pooled rat plasma for 1 minute, 2 minute, 5 minute, 20 minute, 60 minute, 80 minute, 100 minute, 120 minute, 150 minute, and 180 minute. The blood samples at each time point were incubated in triplicate. All the blood samples were centrifuged at 5500 rpm for 10 minutes to obtain plasma samples for analysis. Pooled rat plasma was used as blank for background measurement. All samples were analyzed in duplicate.
All plasma samples with SST fusion protein were diluted 15-fold with pooled rat plasma. For example, to a 10 [iL plasma sample with SST fusion protein was added 140 [iL
pooled rat plasma.
Example 5-2: Preparation of Free SST and SST Fusion Protein Standards (1). Preparation of Free SST standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent buffer to produce 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST fusion protein standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting SST fusion protein Stock (2.42 mg/mL) with diluent buffer to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 5-3: ELISA Assay Procedure 1) All kit components were maintained at room temperature (20-25 C) before analysis.
2) 50 .t.L/well of standard, sample, or positive control solution was added to the kit. Then, 25 i.t.L/well of primary antibody was added into each well except the Blank well.
At last, 25 .t.L/well biotinylated peptide was added into each well except the Blank well.
The immunoplate was incubated for 2 hours at room temperature with shaking at 300-400 rpm.
The wells were emptied and washed three times with 300 [IL washing solution.
After the last wash, the wells were emptied by tapping the strip on an absorbent tissue.
3) The contents of the wells were discarded and each well was washed with 300 0_, of lx ETA assay buffer, discard the buffer was, invert and blot dry plate. Repeat 4 times.
4) Add 100 0_, of SA-HRP solution into each well. Incubate the immunoplate for 1 hour at room temperature with shaking at 300-400 rpm.
5) Wash and blot dry the immunoplate 4 times with lx ETA assay buffer as described above in step 3.
6) Add 100 0_, of TMB substrate solution into each well. Cover the immunoplate to protect from light. Incubate the immunoplate for 1 hour at room temperature with shaking at 300-400 rpm.
7) Add 100 0_, 2N HC1 into each well to stop the reaction. The color in the well should change from blue to yellow. If the color change does not appear to be uniform, gently tap the plate to ensure thorough mixing. Proceed to the next step within 20 minutes.
8) Load the immunoplate onto Plate Reader. Read absorbance O.D. at 450nm.
EXAMPLE 6. DETERMINATION OF IN VIVO PHARMACOKINETIC PROFILE
OF FREE SST and SST FUSION PROTEIN IN RAT
SST and SST fusion protein (SEQ ID NO: 1) were administered at 0.02 and 27.1 mg/kg, respectively, doses via tail vein injection to three and five, respectively, male Sprague Dawley rats to determine the pharmacokinetic profiles and parameters of SST and SST fusion protein, respectively (Table 8 and Table 9). The animals were fasted overnight with free access to water prior to injection, and no negative clinical signs were observed afterwards. SST exhibited rapid pharmacokinetic profile in each of the rats administered with SST (Figure 1), and the calculated Ti/2 was 3.5 minutes (Table 4). SST fusion protein exhibited a bi-phasic pharmacokinetic profile in each of the rats administered with SST
fusion protein (Figure 2), where the average a-phase T1/2 (0-0.5 hour) and 0-phase T1/2 (0.75-4 hours) was calculated as 1.01 hour and 6.14 hour, respectively (Table 4).
The calculated half-life of SST fusion protein was significantly longer than the calculated plasma T1/2 of free SST (3.5 minutes) and reported plasma T1/2 of free SST in rat (< 1 minute;
Reference #1) (Table 4). This set of results indicated the SST fusion protein significantly improves the stability and prolongs the half-life of SST in vivo.
Example 6-1: Preparation of Sample The rat was restrained manually at the designated time points, approximately 300 i.t.L
of blood sample was collected via jugular vein into EDTA-K2 tubes and subsequently centrifuged at 4 C and 1500 g for 10 min to obtain plasma samples.
Example 6-2: Preparation of SST and SST Fusion Protein Standards (1). Preparation of Free SST Standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent buffer to produce 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST Fusion Protein Standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting SST fusion protein (2.42 mg/mL) with pooled rat plasma to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 6-3: ELISA Assay Procedure ELISA assay has been performed as described in Example 5-3.
Table 8. Plasma SST and SST Fusion Protein Concentration after IV injection to Rat SST Dose Dose Sampling Time Mean (ng/mL) Standard Deviation (mg/kg) Route (min) 0.02 IV 0 0 NA
1 7.13 5.0 2 4.95 2.1 4 4.30 NA
6 1.07 0.33 8 2.95 NA
27.1 IV 0.00 0.36 NA
0.05 756393.18 223856.37 0.13 743515.21 233145.74 0.25 634640.70 243150.70 0.50 649841.53 252801.38 0.75 560439.29 183395.09 1.25 480207.30 105178.15 2.00 493399.21 90422.74 3.00 416740.09 98435.97 4.00 366465.56 98751.94 Table 9. Pharmacokinetic Parameters of SST and SST Fusion Protein in Rat after Intravenous Administration SST Fusion Protein SST
PK
Unit Mean SD Mean SD
Parameters AUCo-t mg= h/mL 1960799 427419 29.0 16.0 AUCO-mf mg=h/mL 4730184 1698725 43.8 20.1 AUMCo-t mg-112/mL 3502133 775860 55.9 27.1 AUMCo-nif mg-112/mL 36520105 19055736 173 49.9 MRTw h 7.2 2.0 4.1 0.75 CL mL/kg = min 0.11 0.049 510 234 CL mL/ kg-h 6.53 2.9 NA NA
Vdss L/ kg 0.043 0.0058 2.19 1.3
T] y3 (X);
wherein, each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the fusion protein.
In yet another embodiment, the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker;
ALB is an albumin or its analogues or variants.
In certain embodiments, the nucleotide sequence of the present invention is selected to encode an albumin-somatostatin fusion protein from among, SST-(L)xi-ALB (I);
ALB-(L)xi-SST (11);
[SST-(L)Ayi-ALB (III);
ALB-[(L)xi-SST] yl (IV);
[SST-(L)xi ] yi -ALB - [(L)x2.-SS 1] y2 (V);
[SST-(L)x)] yi -ALB - [(L)x2.-SS 1] y2-(L)3-ALB (VI);
[SST-(L)x)] yi -ALB -[(L)2-SST] y2-(L)3-ALB- [(L)4-S S T] y3 (VII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB (VIII);
ALB-(L)) - [ S S T-(L)x2] yi -ALB - [(L)x3-S S 1] y2-(L)x) -ALB (IX); and ALB-(L)xi-[SST-(L)xflyi-ALB-[(L)x3-SST]y2-(L)xi-ALB-[(L)x4-SST] y3 (X);
wherein, each xl, x2, x3, x4, yl, y2, or y3 is independently zero or an integer selected from 1-10, 1-5 or 1-4, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
Another embodiment of the present invention provides a nucleotide sequence .. encoding an albumin-somatostatin fusion protein, wherein the spacer sequence consists of the sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or ¨GGGGS-.
Another certain embodiment of the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein, wherein the second region (b) encodes a polypeptide having at least 85% sequence identity to SEQ ID NO: 19, albumin or a fragment .. thereof.
One embodiment of the present invention provides a nucleotide sequence encoding an albumin-somatostatin fusion protein, wherein the first region (a) encodes a polypeptide having at least 85% sequence identity to either SEQ ID NOS: 17 or 18, SST-14, SST-28, or a fragment thereof.
The present invention also provides a nucleotide sequence encoding an albumin-somatostatin fusion protein comprising:
(a) a first region comprising a nucleotide sequence containing one or more adjacent repeats of a sequence encoding a human somatostatin peptide;
(b) a second region comprising a nucleotide sequence encoding human serum albumin, or a fragment thereof;
(c) a spacer region comprising a nucleotide sequence encoding a polypeptide of residues in length;
wherein the spacer region is present between the first region and the second region, or or between the first region and another first region;
wherein one or more adjacent repeats of a sequence encoding a human somatostatin peptide encodes either SST-14 or SST-28, as represented by SEQ ID NOS:17 and 18, respectively, or a sequence having at least 85% identity to either of these two sequences; or wherein the spacer sequence consists of the sequence encoding the amino acid sequence represented by SEQ ID NO: 31 or GGGGS or by SEQ ID NO: 30 A(EAAAK)4A;
or wherein the region (a) consists of one or more adjacent repeats of either SST-14 or of SST-28, as represented by SEQ ID NOS: 23 and 24, respectively, or a sequence having at least 85% identity to either of these two sequences.
Furthermore, the present invention provides a polypeptide sequence an albumin-somatostatin fusion protein comprising:
(a) a first region comprising a polypeptide sequence of a somatostatin peptide (which may be a human somatostatin peptide);
(b) a second region comprising a polypeptide sequence of serum albumin (which may be a human serum albumin), or a fragment thereof;
(c) a spacer region comprising a polypeptide of 2-100 residues in length.
The spacer region (c) may be present between region (a) and region (b) or between region (a) and region (a). In addition, the region (a) may comprise one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or sequence having 85% identity to either of these sequences.
Another embodiment of the present invention provides a plasmid construct expressing an albumin-somatostatin fusion protein with any of the fusion protein or polypeptide sequences described above.
Yet another embodiment of the present invention includes a bacterial cell transformed with the plasmid construct described above.
A further embodiment of the present invention includes an isolated and purified albumin-somatostatin fusion protein having the polypeptide sequence described above (e.g., a polypeptide sequence of an albumin-somatostatin fusion protein or the plasmid construct expressing such protein).
Table 1. A non-exclusive list of polypeptide sequences SEQ ID NO: Description SEQ ID NO: 1 55T14-A(EAAAK)4A-HSA-A(EAAAK)4A-55T14 SEQ ID NO: 2 HSA-A(EAAAK)4A-55T14 SEQ ID NO: 3 His6-GGS-HSA-GGGGS-55T14-HSA
SEQ ID NO: 4 His6-GGS-HSA-GGGGS-(55T14-GGGGS)2-HSA
SEQ ID NO: 5 HSA-GGGGS-(55T14-GGGGS)2- HSA
SEQ ID NO: 6 Linker GGGGGGGG
SEQ lD NO: 7 SST14-(GGGGS)3-HSA
SEQ lD NO: 8 SST14-A(EAAAK)4A-HSA
SEQ lD NO: 9 His6-GGS-HSA-GGGGS-55T14 SEQ lD NO: 10 55T14-GGGGS-HSA-GGS-His6 SEQ lD NO: 11 HSA-GGGGS-55T14 SEQ lD NO: 12 55T14-GGGGS-HSA
SEQ lD NO: 13 (55T14-GGGGS)2- HSA
SEQ lD NO: 14 (55T14-GGGGS)4- HSA
SEQ lD NO: 15 HSA-(GGGGS)3-55T14 SEQ lD NO: 16 HSA-(GGGGS)6-55T14 SEQ ID NO: 17 Somatostatin-14 (SST-14) SEQ ID NO: 18 Somatostatin-28 (SST-28) SEQ ID NO: 19 Human Serum Albumin (HSA) SEQ lD NO: 20 MDMRVPAQLLGLLLLWLRGARC (Signal Peptide) SEQ lD NO: 21 Linker APAPAPAPAPAPAPAPAPAP
SEQ lD NO: 22 Linker APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
SEQ lD NO: 30 A(EAAAK)4A peptide SEQ lD NO: 31 GGGGS peptide SEQ ID NO: 32 Linker GGGGSLVPRGSGGGGS
SEQ lD NO: 33 Linker GSGSGS
SEQ lD NO: 34 Linker GGGGSLVPRGSGGGG
SEQ ID NO: 35 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 36 Linker GGSGGHMGSGG
SEQ ID NO: 37 Linker GGSGGSGGSGG
SEQ ID NO: 38 Linker GGSGGHMGSGG
SEQ ID NO: 39 Linker GGSGG
SEQ ID NO: 40 Linker GGGGSLVPRGSGGGGS
SEQ ID NO: 41 Linker GGSGGGGG
SEQ ID NO: 42 Linker GSGSGSGS
SEQ ID NO: 43 Linker GGGSEGGGSEGGGSEGGG
SEQ ID NO: 44 Linker AAGAATAA
SEQ ID NO: 45 Linker GGGGG
SEQ ID NO: 46 Linker GGSSG
SEQ ID NO: 47 Linker GSGGGTGGGSG
SEQ ID NO: 48 Linker GSGSGSGSGGSGGSGGSGGSGGSGGS
For the fusion proteins, e.g., SEQ ID NOs: 1-5, 7-10 and 13-16, it should be noted that these are encoded as pro-proteins with a 22 residue signal peptide (SEQ ID NO: 20).
Somatostatin-Albumin Fusion Proteins The invention encompasses polypeptide constructs wherein the somatostatin moiety is encoded by a nucleotide having at least 85% sequence identity to the nucleotide sequence of endogenous human SST-14 or SST-28 (SEQ ID Nos: 23 and 24, respectively).
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety is encoded by a nucleotide having at least 85% sequence identity to the nucleotide sequence of endogenous human serum albumin (SEQ ID NO: 25). The invention further encompasses polypeptide constructs wherein the human serum albumin moiety is a fragment of the endogenous human serum albumin protein, e.g., where it is encoded by a nucleotide consisting of a subsequence of SEQ ID NO: 25. For example, the human serum albumin fragment optionally includes one or more of the three human serum albumin globular domains, each of which contains two subdomains, denominated subdomain IA, TB, IIA, JIB, IIIA, and IIIB (Dockal, 1999, The Journal Of Biological Chemistry, 274(41):
29303-29310).
The invention also encompasses polypeptide constructs wherein the somatostatin moiety has a polypeptide sequence at least 85% sequence identity, preferably at least 90% to the polypeptide sequence of endogenous SST-14 or SST-28 (SEQ ID NOs:17 and 18, respectively).
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety has a polypeptide sequence at least 85% sequence identity to the polypeptide sequence of mature human serum albumin (SEQ ID NO: 19).
The invention also encompasses a fusion protein comprising a signal peptide, a purification tag (His-6), a first linker, a human serum albumin moiety, a second linker and a somatostatin moiety. In one embodiment, the fusion protein is a polypeptide is represented by SEQ ID NO: 9 or a sequence having 85% sequence identity to the same.
The invention also encompasses a fusion protein comprising a somatostatin moiety, a first linker, a human serum albumin moiety, a second linker, a somatostatin moiety and a purification tag (His-6). In one embodiment, the fusion protein is a polypeptide is represented by SEQ ID NO: 10 or a sequence having 85% sequence identity to the same.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 11) encoding a fusion protein comprising an N-terminal human serum albumin moiety and a C-terminal somatostatin moiety separated by a peptide spacer. The invention further encompasses nucleotide sequences encoding an albumin-somatostatin fusion construct which have 85%
sequence identity to SEQ ID NO: 11.
The invention also encompasses a nucleotide sequence (SEQ ID NO: 12) encoding a fusion protein comprising an N-terminal somatostatin moiety and a C-terminal human serum albumin moiety separated by a peptide spacer. The invention further encompasses nucleotide sequences encoding an albumin-somatostatin fusion construct which have 85%
sequence identity to SEQ ID NO: 12.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more copies of the SST-14 or SST-28 sequence arranged in tandem, i.e., "(SST-14)2" or "(SST-14)3"or "(SST-28)2" or "(SST-28)3", respectively.
Optionally, a linker sequence is included between the two or more tandem somatostatin moieties, and/or a signal peptide sequence is included at the N-terminus of the fusion protein.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more copies of the SST-14 sequence arranged in a way that at least one copy of the SST14 is linked on both sides of albumin, respectively.
Optionally, a linker sequence is included between the two or more tandem somatostatin moieties and between somatostatin and albumin, and/or a signal peptide sequence is included at the N-terminus of the fusion protein. For example, the polypeptide construct may include a signal peptide, two SST-14 moieties separated by a spacer, a second spacer, and an HSA moiety as represented.
Optionally, the construct omits the N-terminal signal peptide.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or three copies of the SST-28 sequence arranged in tandem, i.e., "(SST-28)2" or "(SST-28)3", respectively. Optionally, a linker sequence is included between the two or more tandem somatostatin moieties.
The invention also encompasses polypeptide constructs comprising any of the albumin-somatostatin fusion proteins described in the preceding paragraphs, where the albumin-somatostatin fusion protein has an in vivo half-life longer than the endogenous SST-14 or SST-28 peptides.
The invention also encompasses polypeptide constructs comprising any of the albumin-somatostatin fusion proteins described in the preceding paragraphs, wherein the albumin-somatostatin fusion protein has an approximately equal or a greater binding affinity for a somatostatin receptor compared to endogenous SST-14 or SST-28.
The invention also encompasses albumin-somatostatin fusion proteins comprising an N-terminal albumin moiety as represented by SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID
NO: 2, an internal SST moiety and a C-terminal Albumin moiety as represented by SEQ ID
NO: 7 and SEQ ID NO: 8. Optionally, the N-terminus may further include a signal peptide.
Optionally, one or more of the albumin and SST domains may each be separated by an independently selected linker sequence as represented by SEQ ID NO: 1.
In some embodiments, the SST moiety may comprise a pair or plurality of tandem SST sequences, e.g., (SST-14)2 or (SST-28)3, with or without intervening spacing sequences between the two or more tandem SST repeats. Optionally, one or more purification tag sequences may be included in the sequence between two moieties or at the N or C-terminus in order to assist with purification of the fusion protein. An alternative embodiment includes a pair of SST-14 moieties separated by a spacer, as represented by SEQ ID NO:
4. A further embodiment may omit the purification tag (e.g., His6) as shown by the polypeptide sequence represented by SEQ ID NO: 5.
Somatostatin The somatostatin for use with the present invention may be any somatostatin, its analogue or derivative. It may be a human somatostatin, any other isolated or naturally occurring somatostatin. The SST moiety can be an analogue such as octreotide, lanreotide, pasireotide, seglitide, or vapreotide.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises a somatostatin analog. Preferably, such an analog is suitable for expression, as part of a fusion protein, in a recombinant host cell. It is understood that a suitable somatostatin analog sequence may be used in place of the SST-14 or sequences included in any of the examples disclosed herein.
The invention also encompasses polypeptide constructs wherein the somatostatin moiety comprises two or more tandem repeats of a somatostatin polypeptide sequence e.g., SST-14 or SST-28; SEQ ID NOS: 17 and 18, respectively. Each of the repeated somatostatin polypeptide sequences may be a polypeptide sequence having at least 85%
sequence identity to SST-14 or SST-28. These repeated variant sequences are independently selected, i.e., in some embodiments the repeats are identical, whereas in other embodiments they are unique.
Albumin The albumin for use with the present invention may be any albumin, its analogue or variant. The albumin may be human serum albumin, or any other isolated or naturally occurring albumin.
The invention also encompasses polypeptide constructs wherein the human serum albumin moiety comprises a polypeptide sequence variant with alternative arrangement or number of disulfide bonds due to the presence of additional or fewer cysteine residues than the natural form (SEQ ID NO: 25).
Spacer or Linker As described earlier, a spacer or linker can be used with the present invention. The spacer or linker may be independent of the somatostatin or albumin.
The invention also encompasses polypeptide constructs wherein the peptide spacer of alternatively referred to as a linker, consists of a polypeptide sequence of from about 2 to about 100 amino acid residues in length. The invention further encompasses polypeptide constructs wherein the peptide spacer is from about 2 to about 50 amino acid residues in length, preferably from about 2 to about from 30, or more preferably from about 3 to about 20 amino acid residues in length.
The invention also encompasses polypeptide constructs wherein the peptide spacer (alternatively referred to as a linker) has the polypeptide sequence "GGGGS"
(SEQ ID NO:
31). Polypeptides rich in Gly, Ser or Thr offer special advantages include, but not limited to:
(i) rotational freedom of the polypeptide backbone, so that the adjacent domains are free to move; (ii) enhanced solubility; (iii) resistance to proteolysis. In addition, many natural linkers exhibited alpha-helical structures. The alpha-helical structure is more rigid and stable than Gly rich linker. An empirical rigid linker with the sequence of A(EAAAK)4A
(SEQ ID NO:
30) can be used to separate functional domains. In addition to the role of linking protein domains together, artificial linkers may offer other advantages to the production of fusion proteins, such as improving biological activity, increasing protein expression, and achieving desirable pharmacokinetic profiles.
Table 2. A non-exhaustive list of linker sequences that may be used in the fusion protein constructs of the present invention.
GGGGSLVPRGSGGGGS (SEQ ID NO: 32) GSGSGS (SEQ ID NO: 33) GGGGSLVPRGSGGGG (thrombin proteolytic site is underlined) _ (SEQ ID NO: 34) GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined) _ (SEQ ID NO: 35) GGSGGHMGSGG (SEQ ID NO: 36) GGSGGSGGSGG (SEQ ID NO: 37) GGSGGHMGSGG (SEQ ID NO: 38) GGSGG (SEQ ID NO: 39) GGGGSLVPRGSGGGGS (thrombin proteolytic site is underlined) (SEQ ID NO: 40) GGSGGGGG (SEQ ID NO: 41) GSGSGSGS (SEQ ID NO: 42) GGGSEGGGSEGGGSEGGG (SEQ ID NO: 43) AAGAATAA (SEQ ID NO: 44) GGGGG (SEQ ID NO: 45) GGSSG (SEQ ID NO: 46) GSGGGTGGGSG (SEQ ID NO: 47) GT
GSGSGSGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 48) GGS
GGGGGGGG (SEQ ID NO: 6) A(EAAAK)4A (SEQ ID NO: 20) APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 21) APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP
(SEQ ID NO: 22) Preparation of Somatostatin-Albumin fusion protein An embodiment of the invention provides a method for preparation of the somatostatin-albumin fusion protein. In one embodiment, the somatostatin-albumin fusion protein of the invention is prepared by expressing a vector containing the encoding gene and introducing the vector into a suitable host cell. For example, the fusion protein is obtained by expression of a suitable vector in a host such as yeast. In one embodiment, Pichia pastoris GS115 may be used as a suitable expression host, and the vector is pPIC9K. In particular, mammalian cell lines such as CHO or HEK293 can be used as an expression host.
The invention also encompasses plasmid constructs capable of expressing an albumin somatostatin fusion protein comprising a nucleotide sequence encoding a somatostatin albumin fusion protein as described in any of the preceding paragraphs. For example, suitable plasmid constructs include, but are not limited to, the pcDNA3.1 vector represented by SEQ ID NO: 26 with a DNA sequence encoding any of the albumin-somatostatin fusion proteins disclosed herein ligated into the multiple cloning site of this vector. Other suitable protein expression vectors known in the art may be selected based upon the expression host (e.g., an expression vector with a mammalian promoter system would be suitable for expression in a human cell line whereas a yeast or bacterial expression plasmid would be selected if expression in either of these organisms was desired.
The invention also encompasses a bacterial or yeast protein expression system comprising a bacterial or yeast cell transformed with a plasmid construct comprising a nucleotide sequence that encodes a somatostatin albumin fusion protein, as described in any of the preceding paragraphs. Suitable bacterial strains include, for example, Escherichia coli.
Suitable yeast strains include, for example, Pichia pastoris. An exemplary plasmid construct .. includes pPIC9K (Invitrogen) as represented by SEQ ID NO: 27, with a nucleotide sequence encoding any of the albumin-somatostatin fusion proteins described herein incorporated into the multiple cloning site of the vector.
The invention also encompasses isolated and purified fusion somatostatin fusion proteins having a polypeptide sequences as described in any of the preceding paragraphs.
Table 3. A list of nucleotide sequences in certain embodiments of the invention Nucleotide Sequence Encodes the SEQ ID NO: Description following:
SEQ ID NO: 23 55T14 Somatostatin-14 (SST-14) SEQ ID NO: 24 55T28 Somatostatin-28 (SST-28) Human Serum Albumin SEQ ID NO: 25 Human Serum Albumin mature form (HSA) pcDNA3.1(+) Vector SEQ ID NO: 26 pcDNA3.1(+) Vector mammalian expression vector SEQ ID NO: 27 pPIC9K Vector yeast expression vector SEQ ID NO: 28 GGGGS GGGGS Linker SEQ ID NO: 29 A(EAAAK)4A alpha-helical linker When the SST is a somatostatin analogue, an alternative method known in the field can be employed to prepare the conjugate.
Utility of Somatostatin-Albumin Fusion Protein The fusion protein of the present invention can be used to treat conditions for which somatostatin is art-known to be employed. As such, the invention also encompasses methods of treating cancer in a human subject by administering an isolated and purified albumin-somatostatin fusion protein as described in any of the preceding paragraphs, wherein the cancer is any cancer known to respond to somatostatin treatment, e.g., selected from breast cancer, colorectal cancer, liver cancer, lung cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer and prostate cancer.
The invention also encompasses methods of treating cancer in a human subject by administering a composition containing the fusion protein of the present invention, such as an isolated and purified albumin-somatostatin fusion protein as described in any of the preceding paragraphs. The composition can also include a suitable carrier.
Eleven 55T14-Albumin fusion protein constructs with various linker sequences were designed. Eight of these constructs were made into a fusion gene within a plasmid and produced by HEK 293 transient expression at 100 mL scale. The proteins were collected from the culture media, purified through albumin-based affinity purification, and dialyzed to a storage buffer. These fusion proteins were evaluated for their binding affinity to SSTR2 receptor, and also for cell-based activity in inhibiting cAMP production in a overexpression CHO-Kl cell line. The results of these studies indicated that the length and type of linkers significantly affected the SSTR2 receptor binding affinity, the in-vitro cell-based functional activity, and the fusion protein production yield.
SST-Albumin fusion protein of this invention exhibited a significantly longer serum half-life and/or improved pharmacokinetic profile in solution or in a pharmaceutical composition in vitro and/or in vivo compared to the corresponding unfused, free SST
molecules. The stability of free SST and SST fusion protein was compared in in vitro rat plasma. When incubated in freshly prepared rat plasma at 37 C, free SST and SST fusion protein exhibited degradation half-lives of 33 minutes and 5.5 hours, respectively (Table 4).
In vivo pharmacokinetic profiles were also generated to demonstrate the improved stability of SST fusion protein relative to free SST. Rats administered intravenously with SST showed a calculated T1/2 of 3.5 minutes. On the other hand, rats administered intravenously with SST fusion protein exhibited a bi-phasic pharmacokinetic profile, where the a-phase Ti/2 was 1.01 hour and the 0-phase Ti/2 was 6.14 hour. The calculated half-life of SST fusion protein is significantly longer than the calculated T1/2 of free SST in rat (3.5 .. minutes) and the reported plasma Ti/2 of free SST in rat (< 1 minute;
Reference #1) (Table 4).
Table 4. Calculated Half-life of Free SST and SST Fusion Protein for In Vitro Plasma Stability and In Vivo Rat Model T1/2 In Vitro Plasma Stability In Vivo Rat Pharmacokinetic Free SST 33 minutes 3.5 1.0 minutes less than 1 minutes (Ref. #1) a phase (0-0.5 hr) 1.01 0.60 hr SST Fusion Protein 5.5 hour [3 phase (0.75 - 4 hr) 6.14 1.27 hr Reference #1: Yogesh C. Patel and Thomas Wheatley. In Vivo and in Vitro Plasma Disappearance and Metabolism of Somatostatin-28 and Somatostatin-14 in the Rat.
Endocrinology. Vol. 112, No. 1(1992), pages 220-225.
EXAMPLES
Selected embodiments of the invention will be described in further detail with reference to the following experimental and comparative examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLE 1: EXPRESSION IN MAMMALIAN SYSTEMS
Example 1-1. Recombinant gene synthesis Eight constructs corresponding to the fusion proteins listed in Table 5 were prepared.
First, the gene sequence coding each fusion protein was de novo synthesized and subsequently inserted into the pcDNA3.1 vector.
Example 1-2. Plasmid generation Maxi-prep or Mega-prep was used to generate ¨20 mg of each DNA
Example 1-3. Transfection and protein production (A) Suspension cell method FreeStyleTM 293-F Cells were seeded at 0.55-0.6x106 cells/mL in a flask. After about 24 hours, the cells were seeded in a shake flask at 1.1-1.2x106 cells/mL. DNA
was prepared at 500 g DNA / 80 mL in a FreeStyle medium. Polyethylenimine (PEI) was prepared at 1.8 mL PEI per 80 mL in a FreeStyle medium. DNA was mixed in the FreeStyle medium, and the effective amount of PEI was added to the DNA solution, and the mixture is vortexed incubated for about 15 minutes at room temperature to form a DNA-PEI complex.
An 80 mL
of the incubated DNA-PEI complex is added to a cell culture. About 3 hours later, TC
Yeastolate feed (BD) is added to have the final concentration of 4 gram /
liter of culture.
After about 7-8 days, the medium is harvested by centrifugation.
(B) Adherent cell method About 24 hours before transfection, HEK293 cells were seeded to 50-90 %
confluency in a flask, and complete medium is added. After about 24 hours, cells were washed followed by adding basal medium.
DNA and PEI solutions were prepared by adding DNA to a serum free medium. The PEI solution was added to the DNA solution and incubated for 15 minutes to form DNA-PEI
complex at room temperature.
The PEI/DNA mixture was added to cells, and the mixture incubated for about 4-hours at 37 C. The medium was removed and fresh medium with Glutamine and serum was added, followed by incubating at 37 C for 4 days.
The medium was harvested after about 4 days, by centrifuging to collect the supernatant. The precipitate was replenished with fresh medium with L-Glutamine for another 3-day incubation to repeat the harvesting process.
Example 1-4: Protein Concentration, Ni-NTA Purification and Buffer Exchange The collected medium was concentrated by TFF system (Millipore) to a certain volume depending on purification methods (either continuous chromatography or manual batch purification).
The concentrated proteins was incubated with fresh Ni-NTA resin at about 4 C
in binding buffer and washed with wash buffer using either chromatography or batch system.
The protein was eluted with elute buffer and fractions were collected and concentrated to recover the purified protein. The protein can be further purified using size exclusion chromatography purification.
The buffer of the final eluate can be exchanged by dialysis to a desired buffer.
EXAMPLE 2: YIELDS OF SEVERAL SST-ALBUMIN FUSION PROTEINS
The SST-HSA fusion proteins were all expressed in soluble form with high yield. The length or the nature of the linkers can affect the protein yield and solubility of the fusion proteins. The results indicated that the production yield slightly decreased as the fusion protein constructs became longer and more complex. However, all the constructs exhibited yield for scale up production.
Table 5. SST14-HSA fusion protein expression yield Total Production Sequence ID Design amino MW (kDa) Yield acids (g/L) SST14-A(EAAAK)4A-HSA-SEQ ID NO: 1 657 73.8364 0.26 A(EAAAK)4A-SST14 SEQ ID NO: 2 HSA A(EAAAK)4A-55T14 621 70.1543 0.27 SEQ ID NO: 7 55T14-(GGGGS)3-HSA 614 69.112 0.33 SEQ ID NO: 8 55T14-A(EAAAK)4A-HSA 621 70.1543 0.25 SEQ ID NO: 9 613 69.4874 0.30 SEQ ID NO: 10 613 69.4874 0.41 SEQ ID NO: 15 HSA-(GGGGS)3-55T14 614 69.112 0.28 SEQ ID NO: 16 HSA-(GGGGS)6-55T14 629 70.1119 0.29 EXAMPLE 3: BINDING AFFINITY OF SEVERAL SST-ALBUMIN FUSION
PROTEINS
This assay measures binding of [125I]Somatostatin to human somatostatin sst2 receptors. CHO-Ki cells stably transfected with a plasmid encoding the human somatostatin sst2 receptor are used to prepare membranes in modified HEPES pH 7.4 buffer using standard techniques. A 0.1 mg aliquot of membrane is incubated with 0.03 nM
[125I]Somatostatin and tested fusion proteins for 240 minutes at 25 C. Non-specific binding is estimated in the presence of 1 v.1\4 Somatostatin. Membranes are filtered and washed 3 times and the filters are counted to determine [125I]Somatostatin specifically bound.
The competitive binding study 125I-Tyr-somatostatin versus the fusion proteins demonstrated the following results. The efficiency of the inhibition varied depending on the construct of the fusion proteins. The fusion protein construct (SEQ ID NO: 1) with two alpha-helical linker, A(EAAAK)4A, showed 100 % inhibition of the somatostatin and its receptor interaction. The SEQ ID NO: 1 construct has two somatostatin moiety on both N
and C terminal sides of human serum albumin. The smaller construct with one somatostatin on the C terminal side of human serum albumin linked by the same alpha-helical linker (SEQ
ID NO: 2) showed 96 % inhibition. The same construct with the more flexible GGGGS
linker showed lower inhibition of 82 ¨ 85 % depending on the length. The length of GGGGS
linkers also affected the inhibition. The construct with five amino acid GGGGS
linker (SEQ
ID NO: 9 and SEQ ID NO: 8) showed 57-59 % inhibition whereas the constructs with 15 amino acid (SEQ ID NO: 15) or 30 amino acid GGGGS linkers (SEQ ID NO: 16) showed over 80 %, suggesting that longer than five amino acid GGGGS would be more advantageous to SST function. A more rigid A(EAAAK)4A (a-helical) linker would be more efficient in binding than flexible GGGGS linker. A multiple SST can increase the effective concentration of the ligand for SST receptor binding. The position of Histidine purification tag may not affect the binding. Changing the orientation or position of albumin in the fusion protein may further increase the efficiency of the protein binding.
Table 6. Inhibition of 125I-Tyr1-somatostatin binding on SSTR2 by the fusion proteins Inhibition %
Sequence ID Construct ICso (nM) at 0.1 p,M
SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 100 2.38 A(EAAAK)4A-55T14 SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 96 9.41 SEQ ID NO: 7 55T14-(GGGGS)3-Albumin 70 SEQ ID NO: 8 55T14-A(EAAAK)4A-Albumin 79 SEQ ID NO: 9 His6-GGS-Albumin-GGGGS-55T14 59 SEQ ID NO: 10 55T14-GGGGS-Albumin-His6 57 SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 85 33 SEQ ID NO: 16 Albumin-(GGGGS)6-55T14 82 SEQ ID NO: 17 SST-14 0.0069 EXAMPLE 4. INHIBITION OF SEVERAL SST-ALBUMIN FUSION PROTEINS TO
Human recombinant somatostatin sst2a receptors expressed in CHO-Kl cells were used. Test compound and/or vehicle was incubated with the cells (2 x 105 cells/mL) in incubation buffer for 20 minutes at 37 C. Test compound-induced decrease of cAMP by 50 percent or more (50%) relative to the 10 nM Octreotide response indicated sst2a receptor agonist activity.
The inhibition of the accumulation of cAMP was observed in SST receptor type 2 expressing CHO-Kl cells. The value of EC50 was 260 nM. The constructs with longer linkers (SEQ ID NOS: 1, 15, and 2) exhibited lower EC50 values, which coincided with the binding assay data. The alpha-helical linker appeared to be more efficient in the inhibition of cAMP
production, when Albumin-(GGGGS)3-SST14 and Albumin-(GGGGS)3-SST14 EC50 values were compared.
Table 7. EC50 value for the inhibition of cAMP production ECso values of the Sequence ID Construct inhibition of cAMP
production (nM) SEQ ID NO: 1 55T14-A(EAAAK)4A-Albumin- 5.14 A(EAAAK)4A-55T14 SEQ ID NO: 2 Albumin-A(EAAAK)4A-55T14 17.6 SEQ ID NO: 9 H6-GGGGS-Albumin-GGGGS-55T14 260 SEQ ID NO: 15 Albumin-(GGGGS)3-55T14 23 Octreotide 0.041 EXAMPLE 5. DETERMINATION OF STABILITY OF FREE SST AND SST
FUSION PROTEIN IN RAT PLASMA
Improved stability of SST fusion protein in rat plasma was proven using ELISA.
The test results showed that SST fusion protein (SEQ ID NO: 1) exhibited a degradation half-life of 5.5 hours as opposed to free SST, which showed less than 33 minute half-life when incubated in rat plasma at 37 C (Table 4).
Example 5-1: Preparation of Sample 500 pg/mL of free SST or with 750 ng/mL of SST fusion protein (SEQ ID NO: 1) were incubated in pooled rat plasma for 1 minute, 2 minute, 5 minute, 20 minute, 60 minute, 80 minute, 100 minute, 120 minute, 150 minute, and 180 minute. The blood samples at each time point were incubated in triplicate. All the blood samples were centrifuged at 5500 rpm for 10 minutes to obtain plasma samples for analysis. Pooled rat plasma was used as blank for background measurement. All samples were analyzed in duplicate.
All plasma samples with SST fusion protein were diluted 15-fold with pooled rat plasma. For example, to a 10 [iL plasma sample with SST fusion protein was added 140 [iL
pooled rat plasma.
Example 5-2: Preparation of Free SST and SST Fusion Protein Standards (1). Preparation of Free SST standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent buffer to produce 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST fusion protein standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting SST fusion protein Stock (2.42 mg/mL) with diluent buffer to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 5-3: ELISA Assay Procedure 1) All kit components were maintained at room temperature (20-25 C) before analysis.
2) 50 .t.L/well of standard, sample, or positive control solution was added to the kit. Then, 25 i.t.L/well of primary antibody was added into each well except the Blank well.
At last, 25 .t.L/well biotinylated peptide was added into each well except the Blank well.
The immunoplate was incubated for 2 hours at room temperature with shaking at 300-400 rpm.
The wells were emptied and washed three times with 300 [IL washing solution.
After the last wash, the wells were emptied by tapping the strip on an absorbent tissue.
3) The contents of the wells were discarded and each well was washed with 300 0_, of lx ETA assay buffer, discard the buffer was, invert and blot dry plate. Repeat 4 times.
4) Add 100 0_, of SA-HRP solution into each well. Incubate the immunoplate for 1 hour at room temperature with shaking at 300-400 rpm.
5) Wash and blot dry the immunoplate 4 times with lx ETA assay buffer as described above in step 3.
6) Add 100 0_, of TMB substrate solution into each well. Cover the immunoplate to protect from light. Incubate the immunoplate for 1 hour at room temperature with shaking at 300-400 rpm.
7) Add 100 0_, 2N HC1 into each well to stop the reaction. The color in the well should change from blue to yellow. If the color change does not appear to be uniform, gently tap the plate to ensure thorough mixing. Proceed to the next step within 20 minutes.
8) Load the immunoplate onto Plate Reader. Read absorbance O.D. at 450nm.
EXAMPLE 6. DETERMINATION OF IN VIVO PHARMACOKINETIC PROFILE
OF FREE SST and SST FUSION PROTEIN IN RAT
SST and SST fusion protein (SEQ ID NO: 1) were administered at 0.02 and 27.1 mg/kg, respectively, doses via tail vein injection to three and five, respectively, male Sprague Dawley rats to determine the pharmacokinetic profiles and parameters of SST and SST fusion protein, respectively (Table 8 and Table 9). The animals were fasted overnight with free access to water prior to injection, and no negative clinical signs were observed afterwards. SST exhibited rapid pharmacokinetic profile in each of the rats administered with SST (Figure 1), and the calculated Ti/2 was 3.5 minutes (Table 4). SST fusion protein exhibited a bi-phasic pharmacokinetic profile in each of the rats administered with SST
fusion protein (Figure 2), where the average a-phase T1/2 (0-0.5 hour) and 0-phase T1/2 (0.75-4 hours) was calculated as 1.01 hour and 6.14 hour, respectively (Table 4).
The calculated half-life of SST fusion protein was significantly longer than the calculated plasma T1/2 of free SST (3.5 minutes) and reported plasma T1/2 of free SST in rat (< 1 minute;
Reference #1) (Table 4). This set of results indicated the SST fusion protein significantly improves the stability and prolongs the half-life of SST in vivo.
Example 6-1: Preparation of Sample The rat was restrained manually at the designated time points, approximately 300 i.t.L
of blood sample was collected via jugular vein into EDTA-K2 tubes and subsequently centrifuged at 4 C and 1500 g for 10 min to obtain plasma samples.
Example 6-2: Preparation of SST and SST Fusion Protein Standards (1). Preparation of Free SST Standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting Free SST Stock (1 mg/mL) with diluent buffer to produce 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 ng/mL solutions.
(2). Preparation of SST Fusion Protein Standard Standard curve was prepared by the following procedure. Duplicate standard points were prepared by serially diluting SST fusion protein (2.42 mg/mL) with pooled rat plasma to produce 225, 112.5, 56.2, 28.1, 14.1, 7.03, 3.51 and 1.76 ng/mL solutions.
Example 6-3: ELISA Assay Procedure ELISA assay has been performed as described in Example 5-3.
Table 8. Plasma SST and SST Fusion Protein Concentration after IV injection to Rat SST Dose Dose Sampling Time Mean (ng/mL) Standard Deviation (mg/kg) Route (min) 0.02 IV 0 0 NA
1 7.13 5.0 2 4.95 2.1 4 4.30 NA
6 1.07 0.33 8 2.95 NA
27.1 IV 0.00 0.36 NA
0.05 756393.18 223856.37 0.13 743515.21 233145.74 0.25 634640.70 243150.70 0.50 649841.53 252801.38 0.75 560439.29 183395.09 1.25 480207.30 105178.15 2.00 493399.21 90422.74 3.00 416740.09 98435.97 4.00 366465.56 98751.94 Table 9. Pharmacokinetic Parameters of SST and SST Fusion Protein in Rat after Intravenous Administration SST Fusion Protein SST
PK
Unit Mean SD Mean SD
Parameters AUCo-t mg= h/mL 1960799 427419 29.0 16.0 AUCO-mf mg=h/mL 4730184 1698725 43.8 20.1 AUMCo-t mg-112/mL 3502133 775860 55.9 27.1 AUMCo-nif mg-112/mL 36520105 19055736 173 49.9 MRTw h 7.2 2.0 4.1 0.75 CL mL/kg = min 0.11 0.049 510 234 CL mL/ kg-h 6.53 2.9 NA NA
Vdss L/ kg 0.043 0.0058 2.19 1.3
Claims (21)
1. A fusion protein comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin, its analogue or derivative;
L is a spacer or a linker; and ALB is an albumin, its analogue or variant.
an SST;
an L; and an ALB, wherein, SST is a somatostatin, its analogue or derivative;
L is a spacer or a linker; and ALB is an albumin, its analogue or variant.
2. The fusion protein of claim 1, selected from the group consisting of:
SST-(L)x1-ALB (I);
ALB-(L)x1-SST (II);
[SST-(L)x1]y1-ALB (III);
ALB-[(L)x1-SST] y1 (IV);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2 (V);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2-(L)x3-ALB (VI);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2-(L)x3-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)x1-[SST-(L)x2]y1-ALB (VIII);
ALB-(L)x1- [SST-(L)x2]y1-ALB-[(L)3-SST]y2-(L)x1-ALB (IX); and ALB-(L)x1-[SST-(L)x2]y1-ALB-[(L)x3-SST]y2-(L)x1-ALB-[(L)x4-SST]y3 (X);
wherein, x1, x2, x3, x4, y1, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
SST-(L)x1-ALB (I);
ALB-(L)x1-SST (II);
[SST-(L)x1]y1-ALB (III);
ALB-[(L)x1-SST] y1 (IV);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2 (V);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2-(L)x3-ALB (VI);
[SST-(L)x1]y1-ALB-[(L)x2-SST]y2-(L)x3-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)x1-[SST-(L)x2]y1-ALB (VIII);
ALB-(L)x1- [SST-(L)x2]y1-ALB-[(L)3-SST]y2-(L)x1-ALB (IX); and ALB-(L)x1-[SST-(L)x2]y1-ALB-[(L)x3-SST]y2-(L)x1-ALB-[(L)x4-SST]y3 (X);
wherein, x1, x2, x3, x4, y1, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the nucleotide sequence encoding an albumin-somatostatin fusion protein.
3. The fusion protein of claim 1, wherein the SST is either naturally occurring or synthetically manufactured.
4. The fusion protein of claim 1 wherein the SST comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or a sequence having at least 85% identity to either of these sequences.
5. The fusion protein of claim 1, wherein the SST is SST-14 or SST-28.
6. The fusion protein of claim 1, wherein L is either flexible or alpha helically structured polypeptide linker or spacer.
7. The fusion protein of claim 1, wherein L is a polypeptide having 2-100 amino acids.
8. The fusion protein of claim 6, wherein the polypeptide contains at least one GGGGS, A(EAAAK)4A, (AP) n, wherein n is an integer selected from 10-34, (G)8, (G)5, or any combination thereof.
9. The fusion protein of claim 1, wherein ALB is mammalian serum albumin.
10. The fusion protein of claim 1, wherein the mammalian serum albumin is SEQ ID NO:
25, or a sequence having at least 85 % sequence identity thereto.
25, or a sequence having at least 85 % sequence identity thereto.
11. The fusion protein of claim 2, wherein x1, x2, x3, x4 are each independently an integer selected from 1-5.
12. The fusion protein of claim 2, wherein y1, y2, y3 are each independently an integer selected from 1-5.
13. A nucleotide sequence encoding a polypeptide comprising:
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker; and ALB is an albumin or its analogues or variants.
an SST;
an L; and an ALB, wherein, SST is a somatostatin or its analogues or derivatives;
L is a spacer or a linker; and ALB is an albumin or its analogues or variants.
14. The nucleotide sequence of claim 13, encoding a polypeptide that is selected from the group consisting of, SST-(L)x1-ALB (I);
ALB-(L)x1-SST (II);
[SST-(L)x1] y1-ALB (III);
ALB-[(L)x1-SST] y1 (IV);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2 (V);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2-(L)x3-ALB (VI);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2-(L)x3-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)x1-[SST-(L)x2] y1-ALB (VIII);
ALB-(L)x1- [SST-(L)x2] y1-ALB-[(L)x3-SST] y2-(L)x1-ALB (IX); and ALB-(L)x1-[SST-(L)x2] y1-ALB-[(L)x3-SST] y2-(L)x1-ALB-[(L)x4-SST] y3 (X);
wherein, each of x1, x2, x3, x4, y1, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the polypeptide.
ALB-(L)x1-SST (II);
[SST-(L)x1] y1-ALB (III);
ALB-[(L)x1-SST] y1 (IV);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2 (V);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2-(L)x3-ALB (VI);
[SST-(L)x1] y1-ALB-[(L)x2-SST] y2-(L)x3-ALB-[(L)x4-SST] y3 (VII);
ALB-(L)x1-[SST-(L)x2] y1-ALB (VIII);
ALB-(L)x1- [SST-(L)x2] y1-ALB-[(L)x3-SST] y2-(L)x1-ALB (IX); and ALB-(L)x1-[SST-(L)x2] y1-ALB-[(L)x3-SST] y2-(L)x1-ALB-[(L)x4-SST] y3 (X);
wherein, each of x1, x2, x3, x4, y1, y2, or y3 is independently zero or an integer selected from 1-10, provided that there is at least one L present in the polypeptide.
15. The nucleotide sequence of claim 13, encoding the polypeptide sequence, wherein the SST comprises one or more tandem repeats of a sequence encoding SST-14 or SST-28, represented by SEQ ID NOS: 17 or 18, respectively, or a sequence having at least 85%
identity to either SEQ ID NO: 17 or SEQ ID NO: 18.
identity to either SEQ ID NO: 17 or SEQ ID NO: 18.
16. A plasmid construct expressing an albumin-somatostatin fusion protein comprising the fusion protein of claim 1.
17. A bacterial host cell transformed with the plasmid construct of claim 16.
18. The fusion protein of claim 1 that is isolated and purified.
19. A method of treating a disease or disorder of endocrine release in a human subject by administering an effective amount of a pharmaceutical composition comprising the fusion protein of claim 1, wherein the disease or disorder of endocrine release is a condition that responds to the administration of somatostatin.
20. The method of claim 19, wherein the condition is a cancer selected from the group consisting of breast cancer, colorectal cancer, liver cancer, endocrine cancer, neuroendocrine cancers, pancreatic cancer, prostate cancer, brain cancer and lung cancer.
21. The method of claim 20, wherein the cancer expresses somatostatin receptor type 1, 2, 3, 4 or 5.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562121487P | 2015-02-26 | 2015-02-26 | |
US15/249,346 | 2016-08-26 | ||
US15/249,346 US20170002057A1 (en) | 2015-02-26 | 2016-08-26 | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same |
PCT/US2017/039477 WO2018038803A1 (en) | 2016-08-26 | 2017-06-27 | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3032995A1 true CA3032995A1 (en) | 2018-03-01 |
Family
ID=57847239
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3032995A Abandoned CA3032995A1 (en) | 2015-02-26 | 2017-06-27 | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same |
Country Status (4)
Country | Link |
---|---|
AU (1) | AU2017316199A1 (en) |
CA (1) | CA3032995A1 (en) |
RU (1) | RU2019106796A (en) |
TW (1) | TWI719963B (en) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003059934A2 (en) * | 2001-12-21 | 2003-07-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
-
2016
- 2016-02-26 TW TW105106088A patent/TWI719963B/en not_active IP Right Cessation
-
2017
- 2017-06-27 AU AU2017316199A patent/AU2017316199A1/en not_active Abandoned
- 2017-06-27 CA CA3032995A patent/CA3032995A1/en not_active Abandoned
- 2017-06-27 RU RU2019106796A patent/RU2019106796A/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
AU2017316199A1 (en) | 2019-02-14 |
TWI719963B (en) | 2021-03-01 |
RU2019106796A (en) | 2020-09-28 |
RU2019106796A3 (en) | 2020-11-18 |
TW201634482A (en) | 2016-10-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2858662B1 (en) | Fibroblast growth factor 21 proteins | |
JP2017528419A (en) | MIC-1 fusion protein and use thereof | |
KR102433503B1 (en) | MIC-1 compounds and their use | |
JP6714902B2 (en) | Methods for the expression of peptides and proteins | |
CN113396157A (en) | Insulin analogs with reduced insulin receptor binding affinity | |
US20170334971A1 (en) | Alpha-1-Antitrypsin (A1AT) Fusion Proteins and Uses Thereof | |
US20170002057A1 (en) | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same | |
JP6612360B2 (en) | Fusion protein complex and fusion protein having medicinal action | |
WO2018038803A1 (en) | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same | |
CA3032995A1 (en) | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same | |
KR20080109814A (en) | Polypeptide antagonist | |
US20220348623A1 (en) | Stabilized proteolytically activated growth differentiation factor 11 | |
WO2017214543A1 (en) | Glucagon analogs and methods of use thereof | |
WO2017146738A1 (en) | Compositions containing fusion protein of albumin and analogs thereof, methods for making and using the same | |
KR20120137364A (en) | Igf-i poly (ethylene glycol) conjugates | |
EP2768520A1 (en) | Chemically and thermodynamically stable insulin analogues and improved methods for their production | |
KR100535265B1 (en) | Process for preparation of polypeptides of interest from fusion polypeptides | |
AU2020399194A1 (en) | A method of forming a conjugate of a sulfonamide and a polypeptide |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |
Effective date: 20221229 |
|
FZDE | Discontinued |
Effective date: 20221229 |