US20170081389A1 - Albumin variants and conjugates - Google Patents
Albumin variants and conjugates Download PDFInfo
- Publication number
- US20170081389A1 US20170081389A1 US15/280,733 US201615280733A US2017081389A1 US 20170081389 A1 US20170081389 A1 US 20170081389A1 US 201615280733 A US201615280733 A US 201615280733A US 2017081389 A1 US2017081389 A1 US 2017081389A1
- Authority
- US
- United States
- Prior art keywords
- albumin
- protein
- amino acid
- human
- sequence
- 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
- 102000009027 Albumins Human genes 0.000 title claims abstract description 290
- 108010088751 Albumins Proteins 0.000 title claims abstract description 290
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 94
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 90
- 229920001184 polypeptide Polymers 0.000 claims abstract description 85
- 235000001014 amino acid Nutrition 0.000 claims description 131
- 150000001413 amino acids Chemical class 0.000 claims description 115
- 238000000034 method Methods 0.000 claims description 107
- 235000018417 cysteine Nutrition 0.000 claims description 104
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 79
- 125000000539 amino acid group Chemical group 0.000 claims description 57
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 55
- 238000006467 substitution reaction Methods 0.000 claims description 44
- 238000012217 deletion Methods 0.000 claims description 27
- 230000037430 deletion Effects 0.000 claims description 27
- 238000003780 insertion Methods 0.000 claims description 26
- 230000037431 insertion Effects 0.000 claims description 26
- 230000004075 alteration Effects 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 9
- 230000021615 conjugation Effects 0.000 abstract description 151
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 abstract description 67
- 150000001875 compounds Chemical class 0.000 abstract description 39
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 32
- 230000000975 bioactive effect Effects 0.000 abstract description 31
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 201
- 102000004169 proteins and genes Human genes 0.000 description 174
- 235000018102 proteins Nutrition 0.000 description 167
- 108091006905 Human Serum Albumin Proteins 0.000 description 127
- 102000008100 Human Serum Albumin Human genes 0.000 description 127
- 229940024606 amino acid Drugs 0.000 description 121
- 241000282414 Homo sapiens Species 0.000 description 99
- 210000004027 cell Anatomy 0.000 description 87
- 239000013612 plasmid Substances 0.000 description 80
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 77
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 67
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 65
- -1 L159 in human Chemical class 0.000 description 57
- 150000003573 thiols Chemical class 0.000 description 57
- 239000012634 fragment Substances 0.000 description 55
- 230000014509 gene expression Effects 0.000 description 48
- 102000019034 Chemokines Human genes 0.000 description 47
- 108010012236 Chemokines Proteins 0.000 description 47
- 230000035772 mutation Effects 0.000 description 33
- 230000027455 binding Effects 0.000 description 31
- 230000004927 fusion Effects 0.000 description 31
- 108020004414 DNA Proteins 0.000 description 30
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 26
- 150000001945 cysteines Chemical class 0.000 description 26
- 238000000746 purification Methods 0.000 description 25
- 230000004048 modification Effects 0.000 description 24
- 238000012986 modification Methods 0.000 description 24
- 102000005962 receptors Human genes 0.000 description 24
- 108020003175 receptors Proteins 0.000 description 24
- 239000002105 nanoparticle Substances 0.000 description 23
- 241000894007 species Species 0.000 description 23
- 238000007792 addition Methods 0.000 description 22
- 230000000694 effects Effects 0.000 description 21
- 108020004705 Codon Proteins 0.000 description 19
- 239000003814 drug Substances 0.000 description 19
- 239000003446 ligand Substances 0.000 description 19
- 239000000047 product Substances 0.000 description 19
- 230000008859 change Effects 0.000 description 18
- 108091033319 polynucleotide Proteins 0.000 description 18
- 102000040430 polynucleotide Human genes 0.000 description 18
- 239000002157 polynucleotide Substances 0.000 description 18
- 229940079593 drug Drugs 0.000 description 16
- 239000002502 liposome Substances 0.000 description 15
- 239000011159 matrix material Substances 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 14
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 13
- 108091026890 Coding region Proteins 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 13
- 108091008146 restriction endonucleases Proteins 0.000 description 13
- GANZODCWZFAEGN-UHFFFAOYSA-N 5-mercapto-2-nitro-benzoic acid Chemical compound OC(=O)C1=CC(S)=CC=C1[N+]([O-])=O GANZODCWZFAEGN-UHFFFAOYSA-N 0.000 description 12
- 230000008901 benefit Effects 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 230000001965 increasing effect Effects 0.000 description 11
- 230000009466 transformation Effects 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- 210000005253 yeast cell Anatomy 0.000 description 11
- 206010028980 Neoplasm Diseases 0.000 description 10
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 10
- 210000004899 c-terminal region Anatomy 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 238000004949 mass spectrometry Methods 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 102000001902 CC Chemokines Human genes 0.000 description 9
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- 230000028327 secretion Effects 0.000 description 9
- 235000004400 serine Nutrition 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 8
- 102100036845 C-C motif chemokine 22 Human genes 0.000 description 8
- 108010040471 CC Chemokines Proteins 0.000 description 8
- 108010083701 Chemokine CCL22 Proteins 0.000 description 8
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 8
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 8
- 102000007562 Serum Albumin Human genes 0.000 description 8
- 108010071390 Serum Albumin Proteins 0.000 description 8
- 238000010276 construction Methods 0.000 description 8
- 239000013613 expression plasmid Substances 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 102000037865 fusion proteins Human genes 0.000 description 8
- 108020001507 fusion proteins Proteins 0.000 description 8
- 239000012216 imaging agent Substances 0.000 description 8
- 230000001976 improved effect Effects 0.000 description 8
- 102000054765 polymorphisms of proteins Human genes 0.000 description 8
- 239000002243 precursor Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 8
- 241000283690 Bos taurus Species 0.000 description 7
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- 102000003815 Interleukin-11 Human genes 0.000 description 7
- 108090000177 Interleukin-11 Proteins 0.000 description 7
- 102000004388 Interleukin-4 Human genes 0.000 description 7
- 108090000978 Interleukin-4 Proteins 0.000 description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 102000036693 Thrombopoietin Human genes 0.000 description 7
- 108010041111 Thrombopoietin Proteins 0.000 description 7
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 7
- 235000004279 alanine Nutrition 0.000 description 7
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 230000002538 fungal effect Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 102100034871 C-C motif chemokine 8 Human genes 0.000 description 6
- 101001123540 Candida albicans (strain SC5314 / ATCC MYA-2876) Dolichyl-phosphate-mannose-protein mannosyltransferase 1 Proteins 0.000 description 6
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 6
- 108010055166 Chemokine CCL5 Proteins 0.000 description 6
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 102100029880 Glycodelin Human genes 0.000 description 6
- 101000585553 Homo sapiens Glycodelin Proteins 0.000 description 6
- 108010002386 Interleukin-3 Proteins 0.000 description 6
- 241001138401 Kluyveromyces lactis Species 0.000 description 6
- 206010028347 Muscle twitching Diseases 0.000 description 6
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- 102100036154 Platelet basic protein Human genes 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 239000003431 cross linking reagent Substances 0.000 description 6
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 6
- 239000000032 diagnostic agent Substances 0.000 description 6
- 229940039227 diagnostic agent Drugs 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 229940040731 human interleukin-12 Drugs 0.000 description 6
- 229940096397 interleukin-8 Drugs 0.000 description 6
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 6
- 101150053681 pmt gene Proteins 0.000 description 6
- 108020001568 subdomains Proteins 0.000 description 6
- 108010024976 Asparaginase Proteins 0.000 description 5
- 102000016950 Chemokine CXCL1 Human genes 0.000 description 5
- 108010014419 Chemokine CXCL1 Proteins 0.000 description 5
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 5
- 108010047761 Interferon-alpha Proteins 0.000 description 5
- 102000006992 Interferon-alpha Human genes 0.000 description 5
- 102100039064 Interleukin-3 Human genes 0.000 description 5
- 102000004890 Interleukin-8 Human genes 0.000 description 5
- 108090001007 Interleukin-8 Proteins 0.000 description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 5
- 230000004988 N-glycosylation Effects 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 108010058846 Ovalbumin Proteins 0.000 description 5
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 5
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 5
- 229940092253 ovalbumin Drugs 0.000 description 5
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 230000005030 transcription termination Effects 0.000 description 5
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 5
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 102100021933 C-C motif chemokine 25 Human genes 0.000 description 4
- 101710112540 C-C motif chemokine 25 Proteins 0.000 description 4
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 4
- 241000700199 Cavia porcellus Species 0.000 description 4
- 108010055204 Chemokine CCL8 Proteins 0.000 description 4
- 101000609814 Dictyostelium discoideum Protein disulfide-isomerase 1 Proteins 0.000 description 4
- 102100023688 Eotaxin Human genes 0.000 description 4
- 101710139422 Eotaxin Proteins 0.000 description 4
- 241000283074 Equus asinus Species 0.000 description 4
- 241000283073 Equus caballus Species 0.000 description 4
- 102100020997 Fractalkine Human genes 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 4
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 4
- 101001033233 Homo sapiens Interleukin-10 Proteins 0.000 description 4
- 101001055219 Homo sapiens Interleukin-9 receptor Proteins 0.000 description 4
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 4
- 101001114059 Homo sapiens Protein-arginine deiminase type-1 Proteins 0.000 description 4
- 108010078049 Interferon alpha-2 Proteins 0.000 description 4
- 102000014150 Interferons Human genes 0.000 description 4
- 108010050904 Interferons Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102000000585 Interleukin-9 Human genes 0.000 description 4
- 108010002335 Interleukin-9 Proteins 0.000 description 4
- 102100020880 Kit ligand Human genes 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000004989 O-glycosylation Effects 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- 241000251745 Petromyzon marinus Species 0.000 description 4
- 102000004179 Plasminogen Activator Inhibitor 2 Human genes 0.000 description 4
- 108090000614 Plasminogen Activator Inhibitor 2 Proteins 0.000 description 4
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 4
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 4
- 102100023222 Protein-arginine deiminase type-1 Human genes 0.000 description 4
- 241000282898 Sus scrofa Species 0.000 description 4
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 4
- 239000004473 Threonine Substances 0.000 description 4
- 102000004903 Troponin Human genes 0.000 description 4
- 108090001027 Troponin Proteins 0.000 description 4
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 238000004422 calculation algorithm Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 238000007385 chemical modification Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 229960002949 fluorouracil Drugs 0.000 description 4
- UUVWYPNAQBNQJQ-UHFFFAOYSA-N hexamethylmelamine Chemical compound CN(C)C1=NC(N(C)C)=NC(N(C)C)=N1 UUVWYPNAQBNQJQ-UHFFFAOYSA-N 0.000 description 4
- 102000052620 human IL10 Human genes 0.000 description 4
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 4
- 229940118526 interleukin-9 Drugs 0.000 description 4
- GURKHSYORGJETM-WAQYZQTGSA-N irinotecan hydrochloride (anhydrous) Chemical compound Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 GURKHSYORGJETM-WAQYZQTGSA-N 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical class [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000003248 secreting effect Effects 0.000 description 4
- 239000001488 sodium phosphate Substances 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 235000008521 threonine Nutrition 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 3
- 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 3
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 3
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 3
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 3
- VHRSUDSXCMQTMA-PJHHCJLFSA-N 6alpha-methylprednisolone Chemical compound C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)CO)CC[C@H]21 VHRSUDSXCMQTMA-PJHHCJLFSA-N 0.000 description 3
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 3
- 102000015790 Asparaginase Human genes 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 3
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 3
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 3
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 3
- 108091035707 Consensus sequence Proteins 0.000 description 3
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 108010092160 Dactinomycin Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000003951 Erythropoietin Human genes 0.000 description 3
- 108090000394 Erythropoietin Proteins 0.000 description 3
- 108700039887 Essential Genes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010039471 Fas Ligand Protein Proteins 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 239000004277 Ferrous carbonate Substances 0.000 description 3
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 3
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 3
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 3
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 description 3
- 101000854520 Homo sapiens Fractalkine Proteins 0.000 description 3
- 101000852539 Homo sapiens Importin-5 Proteins 0.000 description 3
- 101000960954 Homo sapiens Interleukin-18 Proteins 0.000 description 3
- 101001033279 Homo sapiens Interleukin-3 Proteins 0.000 description 3
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 3
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 3
- 102100036340 Importin-5 Human genes 0.000 description 3
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 3
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 102000049772 Interleukin-16 Human genes 0.000 description 3
- 101800003050 Interleukin-16 Proteins 0.000 description 3
- 102000015696 Interleukins Human genes 0.000 description 3
- 108010063738 Interleukins Proteins 0.000 description 3
- SHGAZHPCJJPHSC-NUEINMDLSA-N Isotretinoin Chemical compound OC(=O)C=C(C)/C=C/C=C(C)C=CC1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-NUEINMDLSA-N 0.000 description 3
- 241000235058 Komagataella pastoris Species 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 3
- 241000282560 Macaca mulatta Species 0.000 description 3
- 108010064136 Monocyte Chemoattractant Proteins Proteins 0.000 description 3
- 102000014962 Monocyte Chemoattractant Proteins Human genes 0.000 description 3
- 108010016076 Octreotide Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 3
- 108010069873 Patched Receptors Proteins 0.000 description 3
- 102000000017 Patched Receptors Human genes 0.000 description 3
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 3
- 241000282405 Pongo abelii Species 0.000 description 3
- 241000270934 Rana catesbeiana Species 0.000 description 3
- 241000235070 Saccharomyces Species 0.000 description 3
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 108010065729 Troponin I Proteins 0.000 description 3
- 102000013394 Troponin I Human genes 0.000 description 3
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 241000269370 Xenopus <genus> Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 3
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 3
- 150000001412 amines Chemical group 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000003114 blood coagulation factor Substances 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 229960005243 carmustine Drugs 0.000 description 3
- 238000007444 cell Immobilization Methods 0.000 description 3
- 229940044683 chemotherapy drug Drugs 0.000 description 3
- 230000001268 conjugating effect Effects 0.000 description 3
- 108010035886 connective tissue-activating peptide Proteins 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 229940105423 erythropoietin Drugs 0.000 description 3
- 229960005420 etoposide Drugs 0.000 description 3
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 description 3
- 235000008191 folinic acid Nutrition 0.000 description 3
- 239000011672 folinic acid Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 102000043959 human IL18 Human genes 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 102000010681 interleukin-8 receptors Human genes 0.000 description 3
- 229960005280 isotretinoin Drugs 0.000 description 3
- GOTYRUGSSMKFNF-UHFFFAOYSA-N lenalidomide Chemical compound C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O GOTYRUGSSMKFNF-UHFFFAOYSA-N 0.000 description 3
- 150000004668 long chain fatty acids Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 229960004961 mechlorethamine Drugs 0.000 description 3
- RQZAXGRLVPAYTJ-GQFGMJRRSA-N megestrol acetate Chemical compound C1=C(C)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 RQZAXGRLVPAYTJ-GQFGMJRRSA-N 0.000 description 3
- 229960004857 mitomycin Drugs 0.000 description 3
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 3
- XWXYUMMDTVBTOU-UHFFFAOYSA-N nilutamide Chemical compound O=C1C(C)(C)NC(=O)N1C1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 XWXYUMMDTVBTOU-UHFFFAOYSA-N 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 229920000747 poly(lactic acid) Polymers 0.000 description 3
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 3
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 3
- 150000003141 primary amines Chemical class 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 3
- 229960003087 tioguanine Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- 238000000825 ultraviolet detection Methods 0.000 description 3
- 239000004474 valine Substances 0.000 description 3
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine Chemical compound C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-CFWMRBGOSA-N 0.000 description 3
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- BMKDZUISNHGIBY-ZETCQYMHSA-N (+)-dexrazoxane Chemical compound C([C@H](C)N1CC(=O)NC(=O)C1)N1CC(=O)NC(=O)C1 BMKDZUISNHGIBY-ZETCQYMHSA-N 0.000 description 2
- PMJWDPGOWBRILU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCC(C=C1)=CC=C1N1C(=O)C=CC1=O PMJWDPGOWBRILU-UHFFFAOYSA-N 0.000 description 2
- DIYPCWKHSODVAP-UHFFFAOYSA-N 1-[3-(2,5-dioxopyrrol-1-yl)benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 DIYPCWKHSODVAP-UHFFFAOYSA-N 0.000 description 2
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 description 2
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 2
- RTQWWZBSTRGEAV-PKHIMPSTSA-N 2-[[(2s)-2-[bis(carboxymethyl)amino]-3-[4-(methylcarbamoylamino)phenyl]propyl]-[2-[bis(carboxymethyl)amino]propyl]amino]acetic acid Chemical compound CNC(=O)NC1=CC=C(C[C@@H](CN(CC(C)N(CC(O)=O)CC(O)=O)CC(O)=O)N(CC(O)=O)CC(O)=O)C=C1 RTQWWZBSTRGEAV-PKHIMPSTSA-N 0.000 description 2
- 108020005065 3' Flanking Region Proteins 0.000 description 2
- NITXODYAMWZEJY-UHFFFAOYSA-N 3-(pyridin-2-yldisulfanyl)propanehydrazide Chemical compound NNC(=O)CCSSC1=CC=CC=N1 NITXODYAMWZEJY-UHFFFAOYSA-N 0.000 description 2
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 2
- XAUDJQYHKZQPEU-KVQBGUIXSA-N 5-aza-2'-deoxycytidine Chemical compound O=C1N=C(N)N=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XAUDJQYHKZQPEU-KVQBGUIXSA-N 0.000 description 2
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 2
- 102100028814 ATP synthase mitochondrial F1 complex assembly factor 1 Human genes 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 108010077480 Albumin Receptors Proteins 0.000 description 2
- 102100034044 All-trans-retinol dehydrogenase [NAD(+)] ADH1B Human genes 0.000 description 2
- 101710193111 All-trans-retinol dehydrogenase [NAD(+)] ADH4 Proteins 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 2
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 2
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- BFYIZQONLCFLEV-DAELLWKTSA-N Aromasine Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC(=C)C2=C1 BFYIZQONLCFLEV-DAELLWKTSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 2
- 101100434663 Bacillus subtilis (strain 168) fbaA gene Proteins 0.000 description 2
- 102100029516 Basic salivary proline-rich protein 1 Human genes 0.000 description 2
- 108010006654 Bleomycin Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 2
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 2
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 2
- 102100023702 C-C motif chemokine 13 Human genes 0.000 description 2
- 101710112613 C-C motif chemokine 13 Proteins 0.000 description 2
- 102100023703 C-C motif chemokine 15 Human genes 0.000 description 2
- 102100036850 C-C motif chemokine 23 Human genes 0.000 description 2
- 101710098275 C-X-C motif chemokine 10 Proteins 0.000 description 2
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 2
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 description 2
- 102100036153 C-X-C motif chemokine 6 Human genes 0.000 description 2
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 2
- 108010078239 Chemokine CX3CL1 Proteins 0.000 description 2
- 108010014423 Chemokine CXCL6 Proteins 0.000 description 2
- 108010062580 Concanavalin A Proteins 0.000 description 2
- 108010019673 Darbepoetin alfa Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010074604 Epoetin Alfa Proteins 0.000 description 2
- 101150095274 FBA1 gene Proteins 0.000 description 2
- 101150050927 Fcgrt gene Proteins 0.000 description 2
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 2
- 108050003239 Fibroblast growth factor 12 Proteins 0.000 description 2
- 102100028417 Fibroblast growth factor 12 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 description 2
- 102100037665 Fibroblast growth factor 9 Human genes 0.000 description 2
- 108090000367 Fibroblast growth factor 9 Proteins 0.000 description 2
- 108010029961 Filgrastim Proteins 0.000 description 2
- VWUXBMIQPBEWFH-WCCTWKNTSA-N Fulvestrant Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3[C@H](CCCCCCCCCS(=O)CCCC(F)(F)C(F)(F)F)CC2=C1 VWUXBMIQPBEWFH-WCCTWKNTSA-N 0.000 description 2
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 2
- 241000699694 Gerbillinae Species 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 108010069236 Goserelin Proteins 0.000 description 2
- BLCLNMBMMGCOAS-URPVMXJPSA-N Goserelin Chemical compound C([C@@H](C(=O)N[C@H](COC(C)(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(=O)NNC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 BLCLNMBMMGCOAS-URPVMXJPSA-N 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- 101150009006 HIS3 gene Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 101000789413 Homo sapiens ATP synthase mitochondrial F1 complex assembly factor 1 Proteins 0.000 description 2
- 101001125486 Homo sapiens Basic salivary proline-rich protein 1 Proteins 0.000 description 2
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 2
- 101000978381 Homo sapiens C-C motif chemokine 14 Proteins 0.000 description 2
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 description 2
- 101000713081 Homo sapiens C-C motif chemokine 23 Proteins 0.000 description 2
- 101000858088 Homo sapiens C-X-C motif chemokine 10 Proteins 0.000 description 2
- 101000889128 Homo sapiens C-X-C motif chemokine 2 Proteins 0.000 description 2
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 description 2
- 101001033249 Homo sapiens Interleukin-1 beta Proteins 0.000 description 2
- 101001076430 Homo sapiens Interleukin-13 Proteins 0.000 description 2
- 101001055157 Homo sapiens Interleukin-15 Proteins 0.000 description 2
- 101000960969 Homo sapiens Interleukin-5 Proteins 0.000 description 2
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 2
- 101001043807 Homo sapiens Interleukin-7 Proteins 0.000 description 2
- 101001055216 Homo sapiens Interleukin-9 Proteins 0.000 description 2
- 101000664600 Homo sapiens Tripartite motif-containing protein 3 Proteins 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 2
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 2
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- 108090000467 Interferon-beta Proteins 0.000 description 2
- 102000008070 Interferon-gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102000013691 Interleukin-17 Human genes 0.000 description 2
- 108050003558 Interleukin-17 Proteins 0.000 description 2
- 102000004125 Interleukin-1alpha Human genes 0.000 description 2
- 108010082786 Interleukin-1alpha Proteins 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 101150108662 KAR2 gene Proteins 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 2
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 2
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 2
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 2
- 101150007280 LEU2 gene Proteins 0.000 description 2
- 108010000817 Leuprolide Proteins 0.000 description 2
- 229910009891 LiAc Inorganic materials 0.000 description 2
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 2
- 102100026238 Lymphotoxin-alpha Human genes 0.000 description 2
- 108090000362 Lymphotoxin-beta Proteins 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 108010006519 Molecular Chaperones Proteins 0.000 description 2
- 101100346932 Mus musculus Muc1 gene Proteins 0.000 description 2
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 2
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- 241000272140 Naja kaouthia Species 0.000 description 2
- 108010018674 Neurophysins Proteins 0.000 description 2
- 102000002710 Neurophysins Human genes 0.000 description 2
- 101150101654 PSR1 gene Proteins 0.000 description 2
- SHGAZHPCJJPHSC-UHFFFAOYSA-N Panrexin Chemical compound OC(=O)C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-UHFFFAOYSA-N 0.000 description 2
- 241000179547 Pelophylax shqipericus Species 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108010022233 Plasminogen Activator Inhibitor 1 Proteins 0.000 description 2
- 102100039418 Plasminogen activator inhibitor 1 Human genes 0.000 description 2
- 101710195957 Platelet basic protein Proteins 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 2
- XYONNSVDNIRXKZ-UHFFFAOYSA-N S-methyl methanethiosulfonate Chemical compound CSS(C)(=O)=O XYONNSVDNIRXKZ-UHFFFAOYSA-N 0.000 description 2
- 101100386089 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MET17 gene Proteins 0.000 description 2
- 241000277263 Salmo Species 0.000 description 2
- 241000277289 Salmo salar Species 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 102100038803 Somatotropin Human genes 0.000 description 2
- 241000270340 Sphenodon punctatus Species 0.000 description 2
- 108010039445 Stem Cell Factor Proteins 0.000 description 2
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 2
- NAVMQTYZDKMPEU-UHFFFAOYSA-N Targretin Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C(=C)C1=CC=C(C(O)=O)C=C1 NAVMQTYZDKMPEU-UHFFFAOYSA-N 0.000 description 2
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- 108010022394 Threonine synthase Proteins 0.000 description 2
- 102100031372 Thymidine phosphorylase Human genes 0.000 description 2
- 108700023160 Thymidine phosphorylases Proteins 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 102100038798 Tripartite motif-containing protein 3 Human genes 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 102100040247 Tumor necrosis factor Human genes 0.000 description 2
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 description 2
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 2
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 241000235017 Zygosaccharomyces Species 0.000 description 2
- 241000235033 Zygosaccharomyces rouxii Species 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 108700025316 aldesleukin Proteins 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 229960000473 altretamine Drugs 0.000 description 2
- JKOQGQFVAUAYPM-UHFFFAOYSA-N amifostine Chemical compound NCCCNCCSP(O)(O)=O JKOQGQFVAUAYPM-UHFFFAOYSA-N 0.000 description 2
- ROBVIMPUHSLWNV-UHFFFAOYSA-N aminoglutethimide Chemical compound C=1C=C(N)C=CC=1C1(CC)CCC(=O)NC1=O ROBVIMPUHSLWNV-UHFFFAOYSA-N 0.000 description 2
- 229960003437 aminoglutethimide Drugs 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 2
- OTBXOEAOVRKTNQ-UHFFFAOYSA-N anagrelide Chemical compound N1=C2NC(=O)CN2CC2=C(Cl)C(Cl)=CC=C21 OTBXOEAOVRKTNQ-UHFFFAOYSA-N 0.000 description 2
- YBBLVLTVTVSKRW-UHFFFAOYSA-N anastrozole Chemical compound N#CC(C)(C)C1=CC(C(C)(C#N)C)=CC(CN2N=CN=C2)=C1 YBBLVLTVTVSKRW-UHFFFAOYSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229960004405 aprotinin Drugs 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 2
- 229960003272 asparaginase Drugs 0.000 description 2
- 230000003816 axenic effect Effects 0.000 description 2
- DVQHYTBCTGYNNN-UHFFFAOYSA-N azane;cyclobutane-1,1-dicarboxylic acid;platinum Chemical compound N.N.[Pt].OC(=O)C1(C(O)=O)CCC1 DVQHYTBCTGYNNN-UHFFFAOYSA-N 0.000 description 2
- 238000010296 bead milling Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 102000028861 calmodulin binding Human genes 0.000 description 2
- 108091000084 calmodulin binding Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 2
- 230000002016 colloidosmotic effect Effects 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- BGSOJVFOEQLVMH-VWUMJDOOSA-N cortisol phosphate Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)COP(O)(O)=O)[C@@H]4[C@@H]3CCC2=C1 BGSOJVFOEQLVMH-VWUMJDOOSA-N 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 2
- 229960000684 cytarabine Drugs 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 108010017271 denileukin diftitox Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 229960000605 dexrazoxane Drugs 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 102000004419 dihydrofolate reductase Human genes 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 2
- LIQODXNTTZAGID-OCBXBXKTSA-N etoposide phosphate Chemical compound COC1=C(OP(O)(O)=O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 LIQODXNTTZAGID-OCBXBXKTSA-N 0.000 description 2
- 229960000752 etoposide phosphate Drugs 0.000 description 2
- 229940014425 exodus Drugs 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 2
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- YLRFCQOZQXIBAB-RBZZARIASA-N fluoxymesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)C[C@@H]2O YLRFCQOZQXIBAB-RBZZARIASA-N 0.000 description 2
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 2
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 2
- 229960003297 gemtuzumab ozogamicin Drugs 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 102000046438 human CXCL10 Human genes 0.000 description 2
- 102000056003 human IL15 Human genes 0.000 description 2
- 102000052622 human IL7 Human genes 0.000 description 2
- 102000052627 human IL9 Human genes 0.000 description 2
- 102000057041 human TNF Human genes 0.000 description 2
- 102000019207 human interleukin-13 Human genes 0.000 description 2
- 229960001001 ibritumomab tiuxetan Drugs 0.000 description 2
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 2
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000000893 inhibin 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
- 108010093564 inter-alpha-inhibitor Proteins 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 229960003507 interferon alfa-2b Drugs 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 229960001388 interferon-beta Drugs 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- 229940028885 interleukin-4 Drugs 0.000 description 2
- 229940100601 interleukin-6 Drugs 0.000 description 2
- 229940047122 interleukins Drugs 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 2
- HPJKCIUCZWXJDR-UHFFFAOYSA-N letrozole Chemical compound C1=CC(C#N)=CC=C1C(N1N=CN=C1)C1=CC=C(C#N)C=C1 HPJKCIUCZWXJDR-UHFFFAOYSA-N 0.000 description 2
- 229960001691 leucovorin Drugs 0.000 description 2
- RGLRXNKKBLIBQS-XNHQSDQCSA-N leuprolide acetate Chemical compound CC(O)=O.CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 RGLRXNKKBLIBQS-XNHQSDQCSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- AEUKDPKXTPNBNY-XEYRWQBLSA-N mcp 2 Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)C1=CC=CC=C1 AEUKDPKXTPNBNY-XEYRWQBLSA-N 0.000 description 2
- 229960001924 melphalan Drugs 0.000 description 2
- 229960001428 mercaptopurine Drugs 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 229960004584 methylprednisolone Drugs 0.000 description 2
- IXOXBSCIXZEQEQ-UHTZMRCNSA-N nelarabine Chemical compound C1=NC=2C(OC)=NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O IXOXBSCIXZEQEQ-UHTZMRCNSA-N 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 229960002653 nilutamide Drugs 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-M octanoate Chemical compound CCCCCCCC([O-])=O WWZKQHOCKIZLMA-UHFFFAOYSA-M 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- WRUUGTRCQOWXEG-UHFFFAOYSA-N pamidronate Chemical compound NCCC(O)(P(O)(O)=O)P(O)(O)=O WRUUGTRCQOWXEG-UHFFFAOYSA-N 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- WBXPDJSOTKVWSJ-ZDUSSCGKSA-N pemetrexed Chemical compound C=1NC=2NC(N)=NC(=O)C=2C=1CCC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 WBXPDJSOTKVWSJ-ZDUSSCGKSA-N 0.000 description 2
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 2
- 210000001322 periplasm Anatomy 0.000 description 2
- 101150079312 pgk1 gene Proteins 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 229940127557 pharmaceutical product Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000003016 pheromone Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 2
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229940068968 polysorbate 80 Drugs 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- VJZLQIPZNBPASX-OJJGEMKLSA-L prednisolone sodium phosphate Chemical compound [Na+].[Na+].O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)COP([O-])([O-])=O)[C@@H]4[C@@H]3CCC2=C1 VJZLQIPZNBPASX-OJJGEMKLSA-L 0.000 description 2
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 2
- 239000000651 prodrug Substances 0.000 description 2
- 229940002612 prodrug Drugs 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 239000002510 pyrogen Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000012217 radiopharmaceutical Substances 0.000 description 2
- 229940121896 radiopharmaceutical Drugs 0.000 description 2
- 230000002799 radiopharmaceutical effect Effects 0.000 description 2
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 229960004641 rituximab Drugs 0.000 description 2
- MIXCUJKCXRNYFM-UHFFFAOYSA-M sodium;diiodomethanesulfonate;n-propyl-n-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide Chemical compound [Na+].[O-]S(=O)(=O)C(I)I.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl MIXCUJKCXRNYFM-UHFFFAOYSA-M 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 2
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 150000003568 thioethers Chemical class 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 2
- 229960005267 tositumomab Drugs 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000017105 transposition Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- KDQAABAKXDWYSZ-PNYVAJAMSA-N vinblastine sulfate Chemical compound OS(O)(=O)=O.C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 KDQAABAKXDWYSZ-PNYVAJAMSA-N 0.000 description 2
- 229960004528 vincristine Drugs 0.000 description 2
- WAEXFXRVDQXREF-UHFFFAOYSA-N vorinostat Chemical compound ONC(=O)CCCCCCC(=O)NC1=CC=CC=C1 WAEXFXRVDQXREF-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- XRASPMIURGNCCH-UHFFFAOYSA-N zoledronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CN1C=CN=C1 XRASPMIURGNCCH-UHFFFAOYSA-N 0.000 description 2
- TYKASZBHFXBROF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(2,5-dioxopyrrol-1-yl)acetate Chemical compound O=C1CCC(=O)N1OC(=O)CN1C(=O)C=CC1=O TYKASZBHFXBROF-UHFFFAOYSA-N 0.000 description 1
- PMLAAPKPQHEQLF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)C(CCCC)NC(=O)CCSSC1=CC=CC=N1 PMLAAPKPQHEQLF-UHFFFAOYSA-N 0.000 description 1
- FLCQLSRLQIPNLM-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-acetylsulfanylacetate Chemical compound CC(=O)SCC(=O)ON1C(=O)CCC1=O FLCQLSRLQIPNLM-UHFFFAOYSA-N 0.000 description 1
- VRDGQQTWSGDXCU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-iodoacetate Chemical compound ICC(=O)ON1C(=O)CCC1=O VRDGQQTWSGDXCU-UHFFFAOYSA-N 0.000 description 1
- JKHVDAUOODACDU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCN1C(=O)C=CC1=O JKHVDAUOODACDU-UHFFFAOYSA-N 0.000 description 1
- WGMMKWFUXPMTRW-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[(2-bromoacetyl)amino]propanoate Chemical compound BrCC(=O)NCCC(=O)ON1C(=O)CCC1=O WGMMKWFUXPMTRW-UHFFFAOYSA-N 0.000 description 1
- ZRTJVRDXVSDKPX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-acetylsulfanylpropanoate Chemical compound CC(=O)SCCC(=O)ON1C(=O)CCC1=O ZRTJVRDXVSDKPX-UHFFFAOYSA-N 0.000 description 1
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- BQWBEDSJTMWJAE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[(2-iodoacetyl)amino]benzoate Chemical compound C1=CC(NC(=O)CI)=CC=C1C(=O)ON1C(=O)CCC1=O BQWBEDSJTMWJAE-UHFFFAOYSA-N 0.000 description 1
- GKSPIZSKQWTXQG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate Chemical compound C=1C=C(C(=O)ON2C(CCC2=O)=O)C=CC=1C(C)SSC1=CC=CC=N1 GKSPIZSKQWTXQG-UHFFFAOYSA-N 0.000 description 1
- FUOJEDZPVVDXHI-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 5-azido-2-nitrobenzoate Chemical compound [O-][N+](=O)C1=CC=C(N=[N+]=[N-])C=C1C(=O)ON1C(=O)CCC1=O FUOJEDZPVVDXHI-UHFFFAOYSA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 1
- WCMOHMXWOOBVMZ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCN1C(=O)C=CC1=O WCMOHMXWOOBVMZ-UHFFFAOYSA-N 0.000 description 1
- QYEAAMBIUQLHFQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 QYEAAMBIUQLHFQ-UHFFFAOYSA-N 0.000 description 1
- IHVODYOQUSEYJJ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]amino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)C(CC1)CCC1CN1C(=O)C=CC1=O IHVODYOQUSEYJJ-UHFFFAOYSA-N 0.000 description 1
- QDZOEBFLNHCSSF-PFFBOGFISA-N (2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-1-[(2R)-2-amino-5-carbamimidamidopentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-N-[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]pentanediamide Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CCCNC(N)=N)C1=CC=CC=C1 QDZOEBFLNHCSSF-PFFBOGFISA-N 0.000 description 1
- KSDDQEGWVBODMD-OULINLAESA-N (2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-6-amino-2-[[(2S)-4-amino-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-amino-3-sulfanylpropanoyl]amino]-4-carboxybutanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-3-methylbutanoyl]amino]hexanoyl]amino]-4-carboxybutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxybutanoyl]amino]hexanoyl]amino]-4-methylpentanoyl]amino]-3-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-4-oxobutanoyl]amino]-4-oxobutanoyl]amino]hexanoyl]amino]-3-hydroxybutanoyl]amino]-4-carboxybutanoyl]amino]hexanoyl]amino]-4-carboxybutanoyl]amino]-3-methylpentanoyl]amino]-4-methylpentanoic acid Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CS)CC1=CC=CC=C1 KSDDQEGWVBODMD-OULINLAESA-N 0.000 description 1
- NTTZSGBWJWDEKV-ZBRNBAAYSA-N (2s)-2-aminobutanedioic acid;(2s)-2-amino-4-methylsulfanylbutanoic acid Chemical compound CSCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC(O)=O NTTZSGBWJWDEKV-ZBRNBAAYSA-N 0.000 description 1
- NAALWFYYHHJEFQ-ZASNTINBSA-N (2s,5r,6r)-6-[[(2r)-2-[[6-[4-[bis(2-hydroxyethyl)sulfamoyl]phenyl]-2-oxo-1h-pyridine-3-carbonyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound N([C@@H](C(=O)N[C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C=1C=CC(O)=CC=1)C(=O)C(C(N1)=O)=CC=C1C1=CC=C(S(=O)(=O)N(CCO)CCO)C=C1 NAALWFYYHHJEFQ-ZASNTINBSA-N 0.000 description 1
- DDYAPMZTJAYBOF-ZMYDTDHYSA-N (3S)-4-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-4-amino-1-[[(2S,3S)-1-[[(1S)-1-carboxyethyl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]-3-hydroxybutanoyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoic acid Chemical class [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)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)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DDYAPMZTJAYBOF-ZMYDTDHYSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- LTDQGCFMTVHZKP-UHFFFAOYSA-N (4-bromophenyl)-(4,6-dimethoxy-3-methyl-1-benzofuran-2-yl)methanone Chemical compound O1C2=CC(OC)=CC(OC)=C2C(C)=C1C(=O)C1=CC=C(Br)C=C1 LTDQGCFMTVHZKP-UHFFFAOYSA-N 0.000 description 1
- VNTHYLVDGVBPOU-QQYBVWGSSA-N (7s,9s)-9-acetyl-7-[(2r,4s,5s,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 VNTHYLVDGVBPOU-QQYBVWGSSA-N 0.000 description 1
- FPVKHBSQESCIEP-UHFFFAOYSA-N (8S)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol Natural products C1C(O)C(CO)OC1N1C(NC=NCC2O)=C2N=C1 FPVKHBSQESCIEP-UHFFFAOYSA-N 0.000 description 1
- LKJPYSCBVHEWIU-KRWDZBQOSA-N (R)-bicalutamide Chemical compound C([C@@](O)(C)C(=O)NC=1C=C(C(C#N)=CC=1)C(F)(F)F)S(=O)(=O)C1=CC=C(F)C=C1 LKJPYSCBVHEWIU-KRWDZBQOSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- AYDAHOIUHVUJHQ-UHFFFAOYSA-N 1-(3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-yl)pyrrole-2,5-dione Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=CC=2)OC(=O)C1=CC=2N1C(=O)C=CC1=O AYDAHOIUHVUJHQ-UHFFFAOYSA-N 0.000 description 1
- OJQSISYVGFJJBY-UHFFFAOYSA-N 1-(4-isocyanatophenyl)pyrrole-2,5-dione Chemical compound C1=CC(N=C=O)=CC=C1N1C(=O)C=CC1=O OJQSISYVGFJJBY-UHFFFAOYSA-N 0.000 description 1
- RVRLFABOQXZUJX-UHFFFAOYSA-N 1-[1-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1C(C)N1C(=O)C=CC1=O RVRLFABOQXZUJX-UHFFFAOYSA-N 0.000 description 1
- AASYSXRGODIQGY-UHFFFAOYSA-N 1-[1-(2,5-dioxopyrrol-1-yl)hexyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1C(CCCCC)N1C(=O)C=CC1=O AASYSXRGODIQGY-UHFFFAOYSA-N 0.000 description 1
- SGVWDRVQIYUSRA-UHFFFAOYSA-N 1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CCSSCCN1C(=O)C=CC1=O SGVWDRVQIYUSRA-UHFFFAOYSA-N 0.000 description 1
- FERLGYOHRKHQJP-UHFFFAOYSA-N 1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CCOCCOCCN1C(=O)C=CC1=O FERLGYOHRKHQJP-UHFFFAOYSA-N 0.000 description 1
- OYRSKXCXEFLTEY-UHFFFAOYSA-N 1-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethoxy]ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CCOCCOCCOCCN1C(=O)C=CC1=O OYRSKXCXEFLTEY-UHFFFAOYSA-N 0.000 description 1
- WAAXYLYXYLKHJZ-UHFFFAOYSA-N 1-[3-(1-hydroxy-2,5-dioxopyrrolidine-3-carbonyl)phenyl]pyrrole-2,5-dione Chemical compound O=C1N(O)C(=O)CC1C(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 WAAXYLYXYLKHJZ-UHFFFAOYSA-N 0.000 description 1
- VNJBTKQBKFMEHH-UHFFFAOYSA-N 1-[4-(2,5-dioxopyrrol-1-yl)-2,3-dihydroxybutyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CC(O)C(O)CN1C(=O)C=CC1=O VNJBTKQBKFMEHH-UHFFFAOYSA-N 0.000 description 1
- LCZVQHWMSQLWSC-UHFFFAOYSA-N 1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O LCZVQHWMSQLWSC-UHFFFAOYSA-N 0.000 description 1
- CULQNACJHGHAER-UHFFFAOYSA-N 1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 CULQNACJHGHAER-UHFFFAOYSA-N 0.000 description 1
- VHYRLCJMMJQUBY-UHFFFAOYSA-N 1-[4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCC1=CC=C(N2C(C=CC2=O)=O)C=C1 VHYRLCJMMJQUBY-UHFFFAOYSA-N 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- 102100024341 10 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- VHRSUDSXCMQTMA-UHFFFAOYSA-N 11,17-dihydroxy-17-(2-hydroxyacetyl)-6,10,13-trimethyl-7,8,9,11,12,14,15,16-octahydro-6h-cyclopenta[a]phenanthren-3-one Chemical compound CC12C=CC(=O)C=C1C(C)CC1C2C(O)CC2(C)C(O)(C(=O)CO)CCC21 VHRSUDSXCMQTMA-UHFFFAOYSA-N 0.000 description 1
- FUFLCEKSBBHCMO-UHFFFAOYSA-N 11-dehydrocorticosterone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 FUFLCEKSBBHCMO-UHFFFAOYSA-N 0.000 description 1
- NMIZONYLXCOHEF-UHFFFAOYSA-N 1h-imidazole-2-carboxamide Chemical compound NC(=O)C1=NC=CN1 NMIZONYLXCOHEF-UHFFFAOYSA-N 0.000 description 1
- ASNTZYQMIUCEBV-UHFFFAOYSA-N 2,5-dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 ASNTZYQMIUCEBV-UHFFFAOYSA-N 0.000 description 1
- YRJADZYFKNSORZ-UHFFFAOYSA-N 2-[(2-methylphenyl)disulfanyl]pyridine Chemical compound CC1=CC=CC=C1SSC1=CC=CC=N1 YRJADZYFKNSORZ-UHFFFAOYSA-N 0.000 description 1
- QXLQZLBNPTZMRK-UHFFFAOYSA-N 2-[(dimethylamino)methyl]-1-(2,4-dimethylphenyl)prop-2-en-1-one Chemical compound CN(C)CC(=C)C(=O)C1=CC=C(C)C=C1C QXLQZLBNPTZMRK-UHFFFAOYSA-N 0.000 description 1
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- JMUAKWNHKQBPGJ-UHFFFAOYSA-N 3-(pyridin-2-yldisulfanyl)-n-[4-[3-(pyridin-2-yldisulfanyl)propanoylamino]butyl]propanamide Chemical compound C=1C=CC=NC=1SSCCC(=O)NCCCCNC(=O)CCSSC1=CC=CC=N1 JMUAKWNHKQBPGJ-UHFFFAOYSA-N 0.000 description 1
- WUIABRMSWOKTOF-OYALTWQYSA-O 3-[[2-[2-[2-[[(2s,3r)-2-[[(2s,3s,4r)-4-[[(2s,3r)-2-[[6-amino-2-[(1s)-3-amino-1-[[(2s)-2,3-diamino-3-oxopropyl]amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[(2r,3s,4s,5s,6s)-3-[(2r,3s,4s,5r,6r)-4-carbamoyloxy-3,5-dihydroxy-6-(hydroxymethyl)ox Chemical compound OS(O)(=O)=O.N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C WUIABRMSWOKTOF-OYALTWQYSA-O 0.000 description 1
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 102000002627 4-1BB Ligand Human genes 0.000 description 1
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- ZMRMMAOBSFSXLN-UHFFFAOYSA-N 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanehydrazide Chemical compound C1=CC(CCCC(=O)NN)=CC=C1N1C(=O)C=CC1=O ZMRMMAOBSFSXLN-UHFFFAOYSA-N 0.000 description 1
- QTQGHKVYLQBJLO-UHFFFAOYSA-N 4-methylbenzenesulfonate;(4-methyl-1-oxo-1-phenylmethoxypentan-2-yl)azanium Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1.CC(C)CC(N)C(=O)OCC1=CC=CC=C1 QTQGHKVYLQBJLO-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 1
- SHGAZHPCJJPHSC-ZVCIMWCZSA-N 9-cis-retinoic acid Chemical compound OC(=O)/C=C(\C)/C=C/C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-ZVCIMWCZSA-N 0.000 description 1
- 101150016901 ALB1 gene Proteins 0.000 description 1
- 102100027520 ATP synthase mitochondrial F1 complex assembly factor 2 Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 1
- 102100036774 Afamin Human genes 0.000 description 1
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 1
- 102100027211 Albumin Human genes 0.000 description 1
- 101710150350 Albumin-2 Proteins 0.000 description 1
- 108010012934 Albumin-Bound Paclitaxel Proteins 0.000 description 1
- 102100022463 Alpha-1-acid glycoprotein 1 Human genes 0.000 description 1
- 102100022460 Alpha-1-acid glycoprotein 2 Human genes 0.000 description 1
- 241000269331 Ambystoma Species 0.000 description 1
- 241000167989 Ambystoma maculatum Species 0.000 description 1
- 102400000068 Angiostatin Human genes 0.000 description 1
- 108010079709 Angiostatins Proteins 0.000 description 1
- 101710163968 Antistasin Proteins 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 102100029464 Aquaporin-9 Human genes 0.000 description 1
- 101100389688 Arabidopsis thaliana AERO1 gene Proteins 0.000 description 1
- 101100274294 Arabidopsis thaliana CHLD gene Proteins 0.000 description 1
- 101100452478 Arabidopsis thaliana DHAD gene Proteins 0.000 description 1
- 102100031491 Arylsulfatase B Human genes 0.000 description 1
- 101100136076 Aspergillus oryzae (strain ATCC 42149 / RIB 40) pel1 gene Proteins 0.000 description 1
- 101000669426 Aspergillus restrictus Ribonuclease mitogillin Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108050001427 Avidin/streptavidin Proteins 0.000 description 1
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 1
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- NTTIDCCSYIDANP-UHFFFAOYSA-N BCCP Chemical compound BCCP NTTIDCCSYIDANP-UHFFFAOYSA-N 0.000 description 1
- 101150105736 BTT1 gene Proteins 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091071247 Beta family Proteins 0.000 description 1
- 102100030981 Beta-alanine-activating enzyme Human genes 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 101800003265 Beta-thromboglobulin Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 101710201279 Biotin carboxyl carrier protein Proteins 0.000 description 1
- 101710180532 Biotin carboxyl carrier protein of acetyl-CoA carboxylase Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000120506 Bluetongue virus Species 0.000 description 1
- 108010049976 Bone Morphogenetic Protein 5 Proteins 0.000 description 1
- 108010049974 Bone Morphogenetic Protein 6 Proteins 0.000 description 1
- 102000001893 Bone Morphogenetic Protein Receptors Human genes 0.000 description 1
- 108010040422 Bone Morphogenetic Protein Receptors Proteins 0.000 description 1
- 102100028726 Bone morphogenetic protein 10 Human genes 0.000 description 1
- 101710118482 Bone morphogenetic protein 10 Proteins 0.000 description 1
- 102000003928 Bone morphogenetic protein 15 Human genes 0.000 description 1
- 108090000349 Bone morphogenetic protein 15 Proteins 0.000 description 1
- 102100022526 Bone morphogenetic protein 5 Human genes 0.000 description 1
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 description 1
- 102100022545 Bone morphogenetic protein 8B Human genes 0.000 description 1
- 102100031151 C-C chemokine receptor type 2 Human genes 0.000 description 1
- 101710149815 C-C chemokine receptor type 2 Proteins 0.000 description 1
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 1
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 1
- 102100023700 C-C motif chemokine 16 Human genes 0.000 description 1
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 1
- 101710112541 C-C motif chemokine 20 Proteins 0.000 description 1
- 102100031102 C-C motif chemokine 4 Human genes 0.000 description 1
- 101710155834 C-C motif chemokine 7 Proteins 0.000 description 1
- 102100032366 C-C motif chemokine 7 Human genes 0.000 description 1
- 101710155833 C-C motif chemokine 8 Proteins 0.000 description 1
- 102100028990 C-X-C chemokine receptor type 3 Human genes 0.000 description 1
- 102100039398 C-X-C motif chemokine 2 Human genes 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 108700012434 CCL3 Proteins 0.000 description 1
- 102000004497 CCR2 Receptors Human genes 0.000 description 1
- 108010017312 CCR2 Receptors Proteins 0.000 description 1
- 108010046080 CD27 Ligand Proteins 0.000 description 1
- 102000007499 CD27 Ligand Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102000004634 CD30 Ligand Human genes 0.000 description 1
- 108010017987 CD30 Ligand Proteins 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 101150085381 CDC19 gene Proteins 0.000 description 1
- 108050006947 CXC Chemokine Proteins 0.000 description 1
- 102000019388 CXC chemokine Human genes 0.000 description 1
- 108010055172 CXC chemokine Mig Proteins 0.000 description 1
- 102000010919 CXC chemokine receptor 3 Human genes 0.000 description 1
- 108010061300 CXCR3 Receptors Proteins 0.000 description 1
- 101150004010 CXCR3 gene Proteins 0.000 description 1
- 101100054773 Caenorhabditis elegans act-2 gene Proteins 0.000 description 1
- 101100161935 Caenorhabditis elegans act-4 gene Proteins 0.000 description 1
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 description 1
- 101100369802 Caenorhabditis elegans tim-1 gene Proteins 0.000 description 1
- 102400000113 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 102100033620 Calponin-1 Human genes 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241000282461 Canis lupus Species 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 241000283705 Capra hircus Species 0.000 description 1
- OKTJSMMVPCPJKN-NJFSPNSNSA-N Carbon-14 Chemical compound [14C] OKTJSMMVPCPJKN-NJFSPNSNSA-N 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 102100034663 Caseinolytic peptidase B protein homolog Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010059013 Chaperonin 10 Proteins 0.000 description 1
- 108010058432 Chaperonin 60 Proteins 0.000 description 1
- 108010082161 Chemokine CCL19 Proteins 0.000 description 1
- 102000003805 Chemokine CCL19 Human genes 0.000 description 1
- 102000000013 Chemokine CCL3 Human genes 0.000 description 1
- 102000001327 Chemokine CCL5 Human genes 0.000 description 1
- 102000006579 Chemokine CXCL10 Human genes 0.000 description 1
- 108010008978 Chemokine CXCL10 Proteins 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 102000003914 Cholinesterases Human genes 0.000 description 1
- 108090000322 Cholinesterases Proteins 0.000 description 1
- VYZAMTAEIAYCRO-BJUDXGSMSA-N Chromium-51 Chemical compound [51Cr] VYZAMTAEIAYCRO-BJUDXGSMSA-N 0.000 description 1
- 206010061764 Chromosomal deletion Diseases 0.000 description 1
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 1
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 1
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 102100023804 Coagulation factor VII Human genes 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 241000272201 Columbiformes Species 0.000 description 1
- 108010028773 Complement C5 Proteins 0.000 description 1
- 102100031506 Complement C5 Human genes 0.000 description 1
- 102400000498 Connective tissue-activating peptide III Human genes 0.000 description 1
- MFYSYFVPBJMHGN-ZPOLXVRWSA-N Cortisone Chemical compound O=C1CC[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 MFYSYFVPBJMHGN-ZPOLXVRWSA-N 0.000 description 1
- MFYSYFVPBJMHGN-UHFFFAOYSA-N Cortisone Natural products O=C1CCC2(C)C3C(=O)CC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 MFYSYFVPBJMHGN-UHFFFAOYSA-N 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000484025 Cuniculus Species 0.000 description 1
- 102100021906 Cyclin-O Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101150007692 DOA4 gene Proteins 0.000 description 1
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 description 1
- WEAHRLBPCANXCN-UHFFFAOYSA-N Daunomycin Natural products CCC1(O)CC(OC2CC(N)C(O)C(C)O2)c3cc4C(=O)c5c(OC)cccc5C(=O)c4c(O)c3C1 WEAHRLBPCANXCN-UHFFFAOYSA-N 0.000 description 1
- GUGHGUXZJWAIAS-QQYBVWGSSA-N Daunorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 GUGHGUXZJWAIAS-QQYBVWGSSA-N 0.000 description 1
- 108010049207 Death Domain Receptors Proteins 0.000 description 1
- 102000009058 Death Domain Receptors Human genes 0.000 description 1
- 102100030438 Derlin-1 Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102100032788 Dimethylaniline monooxygenase [N-oxide-forming] 1 Human genes 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000251475 Dipnoi Species 0.000 description 1
- 102100035966 DnaJ homolog subfamily A member 2 Human genes 0.000 description 1
- 101100120663 Drosophila melanogaster fs(1)h gene Proteins 0.000 description 1
- 102100023431 E3 ubiquitin-protein ligase TRIM21 Human genes 0.000 description 1
- 102100028890 E3 ubiquitin-protein ligase synoviolin Human genes 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 101150005287 EPS1 gene Proteins 0.000 description 1
- 101150047030 ERO1 gene Proteins 0.000 description 1
- 101150050883 ERV2 gene Proteins 0.000 description 1
- 102100040897 Embryonic growth/differentiation factor 1 Human genes 0.000 description 1
- 102400001047 Endostatin Human genes 0.000 description 1
- 108010079505 Endostatins Proteins 0.000 description 1
- 108010032976 Enfuvirtide Proteins 0.000 description 1
- 101000889812 Enterobacteria phage T4 Endonuclease Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241001337814 Erysiphe glycines Species 0.000 description 1
- 101001056496 Escherichia coli (strain K12) Enterobactin exporter EntS Proteins 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 101150021185 FGF gene Proteins 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010023321 Factor VII Proteins 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 108010014173 Factor X Proteins 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 102100035290 Fibroblast growth factor 13 Human genes 0.000 description 1
- 108050002072 Fibroblast growth factor 16 Proteins 0.000 description 1
- 102100035307 Fibroblast growth factor 16 Human genes 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000378 Fibroblast growth factor 3 Proteins 0.000 description 1
- 102100028043 Fibroblast growth factor 3 Human genes 0.000 description 1
- 108090000380 Fibroblast growth factor 5 Proteins 0.000 description 1
- 102100028073 Fibroblast growth factor 5 Human genes 0.000 description 1
- 108090000382 Fibroblast growth factor 6 Proteins 0.000 description 1
- 102100028075 Fibroblast growth factor 6 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000368 Fibroblast growth factor 8 Proteins 0.000 description 1
- 102100037680 Fibroblast growth factor 8 Human genes 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- MPJKWIXIYCLVCU-UHFFFAOYSA-N Folinic acid Natural products NC1=NC2=C(N(C=O)C(CNc3ccc(cc3)C(=O)NC(CCC(=O)O)CC(=O)O)CN2)C(=O)N1 MPJKWIXIYCLVCU-UHFFFAOYSA-N 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 102100040977 Follitropin subunit beta Human genes 0.000 description 1
- 101710203050 Follitropin subunit beta Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 101150038242 GAL10 gene Proteins 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102000000805 Galectin 4 Human genes 0.000 description 1
- GYHNNYVSQQEPJS-OIOBTWANSA-N Gallium-67 Chemical compound [67Ga] GYHNNYVSQQEPJS-OIOBTWANSA-N 0.000 description 1
- 101100164983 Gallus gallus ATXN3 gene Proteins 0.000 description 1
- 108700004714 Gelonium multiflorum GEL Proteins 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108010088406 Glucagon-Like Peptides Proteins 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- 102000030595 Glucokinase Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 102100031132 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 102000004366 Glucosidases Human genes 0.000 description 1
- 108010056771 Glucosidases Proteins 0.000 description 1
- 108010017544 Glucosylceramidase Proteins 0.000 description 1
- 102000004547 Glucosylceramidase Human genes 0.000 description 1
- 102100036669 Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Human genes 0.000 description 1
- 101710170453 Glycoprotein 55 Proteins 0.000 description 1
- 102100040796 Glycoprotein hormones alpha chain Human genes 0.000 description 1
- 102100025326 Golgin-45 Human genes 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 108010090296 Growth Differentiation Factor 1 Proteins 0.000 description 1
- 108010090290 Growth Differentiation Factor 2 Proteins 0.000 description 1
- 102100040892 Growth/differentiation factor 2 Human genes 0.000 description 1
- 102100030488 HEAT repeat-containing protein 6 Human genes 0.000 description 1
- 101150069554 HIS4 gene Proteins 0.000 description 1
- 108010010369 HIV Protease Proteins 0.000 description 1
- 101150068227 HSP104 gene Proteins 0.000 description 1
- 101150079981 HSP150 gene Proteins 0.000 description 1
- 101150007068 HSP81-1 gene Proteins 0.000 description 1
- 101150087422 HSP82 gene Proteins 0.000 description 1
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 102000007625 Hirudins Human genes 0.000 description 1
- 108010007267 Hirudins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000936108 Homo sapiens ATP synthase mitochondrial F1 complex assembly factor 2 Proteins 0.000 description 1
- 101000928239 Homo sapiens Afamin Proteins 0.000 description 1
- 101000678195 Homo sapiens Alpha-1-acid glycoprotein 1 Proteins 0.000 description 1
- 101000678191 Homo sapiens Alpha-1-acid glycoprotein 2 Proteins 0.000 description 1
- 101000771413 Homo sapiens Aquaporin-9 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101100122513 Homo sapiens BLZF1 gene Proteins 0.000 description 1
- 101000773364 Homo sapiens Beta-alanine-activating enzyme Proteins 0.000 description 1
- 101000899368 Homo sapiens Bone morphogenetic protein 8B Proteins 0.000 description 1
- 101000713104 Homo sapiens C-C motif chemokine 1 Proteins 0.000 description 1
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 1
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 1
- 101000713083 Homo sapiens C-C motif chemokine 22 Proteins 0.000 description 1
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 description 1
- 101000916050 Homo sapiens C-X-C chemokine receptor type 3 Proteins 0.000 description 1
- 101100382876 Homo sapiens CCL16 gene Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000946436 Homo sapiens Caseinolytic peptidase B protein homolog Proteins 0.000 description 1
- 101000897441 Homo sapiens Cyclin-O Proteins 0.000 description 1
- 101000842611 Homo sapiens Derlin-1 Proteins 0.000 description 1
- 101000931210 Homo sapiens DnaJ homolog subfamily A member 2 Proteins 0.000 description 1
- 101000685877 Homo sapiens E3 ubiquitin-protein ligase TRIM21 Proteins 0.000 description 1
- 101000838967 Homo sapiens E3 ubiquitin-protein ligase synoviolin Proteins 0.000 description 1
- 101000921370 Homo sapiens Elongation of very long chain fatty acids protein 1 Proteins 0.000 description 1
- 101001060274 Homo sapiens Fibroblast growth factor 4 Proteins 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 101001072574 Homo sapiens Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Proteins 0.000 description 1
- 101001038874 Homo sapiens Glycoprotein hormones alpha chain Proteins 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 description 1
- 101000990566 Homo sapiens HEAT repeat-containing protein 6 Proteins 0.000 description 1
- 101000777670 Homo sapiens Hsp90 co-chaperone Cdc37 Proteins 0.000 description 1
- 101000960952 Homo sapiens Interleukin-1 receptor accessory protein Proteins 0.000 description 1
- 101001076407 Homo sapiens Interleukin-1 receptor antagonist protein Proteins 0.000 description 1
- 101001076422 Homo sapiens Interleukin-1 receptor type 2 Proteins 0.000 description 1
- 101001010568 Homo sapiens Interleukin-11 Proteins 0.000 description 1
- 101001019598 Homo sapiens Interleukin-17 receptor A Proteins 0.000 description 1
- 101001040964 Homo sapiens Interleukin-36 receptor antagonist protein Proteins 0.000 description 1
- 101001033312 Homo sapiens Interleukin-4 receptor subunit alpha Proteins 0.000 description 1
- 101000718476 Homo sapiens L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase Proteins 0.000 description 1
- 101000764535 Homo sapiens Lymphotoxin-alpha Proteins 0.000 description 1
- 101001018100 Homo sapiens Lysozyme C Proteins 0.000 description 1
- 101000957559 Homo sapiens Matrin-3 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101001030243 Homo sapiens Myosin-7 Proteins 0.000 description 1
- 101000836873 Homo sapiens Nucleotide exchange factor SIL1 Proteins 0.000 description 1
- 101001099381 Homo sapiens Peroxisomal biogenesis factor 19 Proteins 0.000 description 1
- 101000801684 Homo sapiens Phospholipid-transporting ATPase ABCA1 Proteins 0.000 description 1
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 1
- 101000609255 Homo sapiens Plasminogen activator inhibitor 1 Proteins 0.000 description 1
- 101000605345 Homo sapiens Prefoldin subunit 1 Proteins 0.000 description 1
- 101000742143 Homo sapiens Prenylated Rab acceptor protein 1 Proteins 0.000 description 1
- 101001055149 Homo sapiens Pro-interleukin-16 Proteins 0.000 description 1
- 101000685275 Homo sapiens Protein sel-1 homolog 1 Proteins 0.000 description 1
- 101000797990 Homo sapiens Putative activator of 90 kDa heat shock protein ATPase homolog 2 Proteins 0.000 description 1
- 101001109419 Homo sapiens RNA-binding protein NOB1 Proteins 0.000 description 1
- 101000685886 Homo sapiens RNA-binding protein RO60 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000824890 Homo sapiens SOSS complex subunit B2 Proteins 0.000 description 1
- 101000642268 Homo sapiens Speckle-type POZ protein Proteins 0.000 description 1
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 1
- 101000837443 Homo sapiens T-complex protein 1 subunit beta Proteins 0.000 description 1
- 101000653567 Homo sapiens T-complex protein 1 subunit delta Proteins 0.000 description 1
- 101000653663 Homo sapiens T-complex protein 1 subunit epsilon Proteins 0.000 description 1
- 101000713879 Homo sapiens T-complex protein 1 subunit eta Proteins 0.000 description 1
- 101000595467 Homo sapiens T-complex protein 1 subunit gamma Proteins 0.000 description 1
- 101000835696 Homo sapiens T-complex protein 1 subunit theta Proteins 0.000 description 1
- 101000653469 Homo sapiens T-complex protein 1 subunit zeta Proteins 0.000 description 1
- 101000845188 Homo sapiens Tetratricopeptide repeat protein 4 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 1
- 101000801228 Homo sapiens Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 1
- 101000597785 Homo sapiens Tumor necrosis factor receptor superfamily member 6B Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 101000920026 Homo sapiens Tumor necrosis factor receptor superfamily member EDAR Proteins 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 101000759555 Homo sapiens Zinc finger and BTB domain-containing protein 7C Proteins 0.000 description 1
- 101150050258 Hsp26 gene Proteins 0.000 description 1
- 101150028525 Hsp83 gene Proteins 0.000 description 1
- 102100031568 Hsp90 co-chaperone Cdc37 Human genes 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 108010016183 Human immunodeficiency virus 1 p16 protease Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 1
- OUBORTRIKPEZMG-UHFFFAOYSA-N INT-2 Chemical compound Nc1c(ncn1-c1ccc(F)cc1)C(=N)C#N OUBORTRIKPEZMG-UHFFFAOYSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 101710096421 Iduronate 2-sulfatase Proteins 0.000 description 1
- 102100029199 Iduronate 2-sulfatase Human genes 0.000 description 1
- 108010003381 Iduronidase Proteins 0.000 description 1
- 102000004627 Iduronidase Human genes 0.000 description 1
- 241001562081 Ikeda Species 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000014429 Insulin-like growth factor Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 102000004551 Interleukin-10 Receptors Human genes 0.000 description 1
- 108010017550 Interleukin-10 Receptors Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000004560 Interleukin-12 Receptors Human genes 0.000 description 1
- 108010017515 Interleukin-12 Receptors Proteins 0.000 description 1
- 102000014154 Interleukin-12 Subunit p35 Human genes 0.000 description 1
- 108010011301 Interleukin-12 Subunit p35 Proteins 0.000 description 1
- 102000003816 Interleukin-13 Human genes 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 1
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 1
- 102000004554 Interleukin-17 Receptors Human genes 0.000 description 1
- 108010017525 Interleukin-17 Receptors Proteins 0.000 description 1
- 102000003810 Interleukin-18 Human genes 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 102100039879 Interleukin-19 Human genes 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- 102000010790 Interleukin-3 Receptors Human genes 0.000 description 1
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 1
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 1
- 102000010782 Interleukin-7 Receptors Human genes 0.000 description 1
- 108010038498 Interleukin-7 Receptors Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- ZCYVEMRRCGMTRW-AHCXROLUSA-N Iodine-123 Chemical compound [123I] ZCYVEMRRCGMTRW-AHCXROLUSA-N 0.000 description 1
- XEEYBQQBJWHFJM-AKLPVKDBSA-N Iron-59 Chemical compound [59Fe] XEEYBQQBJWHFJM-AKLPVKDBSA-N 0.000 description 1
- 229940124726 Japanese encephalitis vaccine Drugs 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 102100026384 L-aminoadipate-semialdehyde dehydrogenase-phosphopantetheinyl transferase Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000002067 L01XE06 - Dasatinib Substances 0.000 description 1
- 101150096459 LHS1 gene Proteins 0.000 description 1
- 241000235031 Lachancea fermentati Species 0.000 description 1
- 101000844802 Lacticaseibacillus rhamnosus Teichoic acid D-alanyltransferase Proteins 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 102000008121 Latent TGF-beta Binding Proteins Human genes 0.000 description 1
- 108010049807 Latent TGF-beta Binding Proteins Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 241000209499 Lemna Species 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 102100035304 Lymphotactin Human genes 0.000 description 1
- 102100026894 Lymphotoxin-beta Human genes 0.000 description 1
- 101150014737 MADS1 gene Proteins 0.000 description 1
- 241000282553 Macaca Species 0.000 description 1
- 108010058398 Macrophage Colony-Stimulating Factor Receptor Proteins 0.000 description 1
- 101710091437 Major capsid protein 2 Proteins 0.000 description 1
- 108010087568 Mannosyltransferases Proteins 0.000 description 1
- 102000006722 Mannosyltransferases Human genes 0.000 description 1
- 101710084218 Master replication protein Proteins 0.000 description 1
- 101710140452 Mating factor alpha-1 Proteins 0.000 description 1
- 102100038645 Matrin-3 Human genes 0.000 description 1
- 241000288145 Meleagris Species 0.000 description 1
- 108010037274 Member 9 Tumor Necrosis Factor Receptor Superfamily Proteins 0.000 description 1
- 102000011769 Member 9 Tumor Necrosis Factor Receptor Superfamily Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699684 Meriones unguiculatus Species 0.000 description 1
- XOGTZOOQQBDUSI-UHFFFAOYSA-M Mesna Chemical compound [Na+].[O-]S(=O)(=O)CCS XOGTZOOQQBDUSI-UHFFFAOYSA-M 0.000 description 1
- FQISKWAFAHGMGT-SGJOWKDISA-M Methylprednisolone sodium succinate Chemical compound [Na+].C([C@@]12C)=CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2[C@@H](O)C[C@]2(C)[C@@](O)(C(=O)COC(=O)CCC([O-])=O)CC[C@H]21 FQISKWAFAHGMGT-SGJOWKDISA-M 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 108090000143 Mouse Proteins Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100434646 Mus musculus Alb gene Proteins 0.000 description 1
- 101000930477 Mus musculus Albumin Proteins 0.000 description 1
- 101000899367 Mus musculus Bone morphogenetic protein 8A Proteins 0.000 description 1
- 101000946797 Mus musculus C-C motif chemokine 9 Proteins 0.000 description 1
- 101100219997 Mus musculus Ccr1 gene Proteins 0.000 description 1
- 101000878182 Mus musculus Fibroblast growth factor 15 Proteins 0.000 description 1
- 206010028289 Muscle atrophy Diseases 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 102100030856 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 102100038934 Myosin-7 Human genes 0.000 description 1
- 108010027520 N-Acetylgalactosamine-4-Sulfatase Proteins 0.000 description 1
- LKJPYSCBVHEWIU-UHFFFAOYSA-N N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfonyl]-2-hydroxy-2-methylpropanamide Chemical compound C=1C=C(C#N)C(C(F)(F)F)=CC=1NC(=O)C(O)(C)CS(=O)(=O)C1=CC=C(F)C=C1 LKJPYSCBVHEWIU-UHFFFAOYSA-N 0.000 description 1
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethyl-succinimide Natural products CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 108020001621 Natriuretic Peptide Proteins 0.000 description 1
- 102000004571 Natriuretic peptide Human genes 0.000 description 1
- 241000251469 Neoceratodus Species 0.000 description 1
- 241000251470 Neoceratodus forsteri Species 0.000 description 1
- 102100028762 Neuropilin-1 Human genes 0.000 description 1
- 108090000772 Neuropilin-1 Proteins 0.000 description 1
- 102100028492 Neuropilin-2 Human genes 0.000 description 1
- 108090000770 Neuropilin-2 Proteins 0.000 description 1
- 101100234604 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) ace-8 gene Proteins 0.000 description 1
- 101100015882 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) grpe gene Proteins 0.000 description 1
- 101100020663 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) ppm-1 gene Proteins 0.000 description 1
- 108090000742 Neurotrophin 3 Proteins 0.000 description 1
- 102000004230 Neurotrophin 3 Human genes 0.000 description 1
- 102000003683 Neurotrophin-4 Human genes 0.000 description 1
- 108090000099 Neurotrophin-4 Proteins 0.000 description 1
- QJGQUHMNIGDVPM-BJUDXGSMSA-N Nitrogen-13 Chemical compound [13N] QJGQUHMNIGDVPM-BJUDXGSMSA-N 0.000 description 1
- 108010041199 Nogo Receptor 1 Proteins 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 102100027096 Nucleotide exchange factor SIL1 Human genes 0.000 description 1
- 241001195348 Nusa Species 0.000 description 1
- JHDRCSNFUVEROF-UHFFFAOYSA-N O=C1CC(S(O)(=O)=O)C(=O)N1OC(=O)C(CCCC)NC(=O)CCSSC1=CC=CC=N1 Chemical compound O=C1CC(S(O)(=O)=O)C(=O)N1OC(=O)C(CCCC)NC(=O)CCSSC1=CC=CC=N1 JHDRCSNFUVEROF-UHFFFAOYSA-N 0.000 description 1
- 108010042215 OX40 Ligand Proteins 0.000 description 1
- 102000004473 OX40 Ligand Human genes 0.000 description 1
- 241000209094 Oryza Species 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 101000930455 Ovis aries Albumin Proteins 0.000 description 1
- 102400000050 Oxytocin Human genes 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 101150101848 PMA1 gene Proteins 0.000 description 1
- 101150093629 PYK1 gene Proteins 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 101710112083 Para-Rep C1 Proteins 0.000 description 1
- 101710112078 Para-Rep C2 Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102000007497 Patched-2 Receptor Human genes 0.000 description 1
- 108010071083 Patched-2 Receptor Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010067162 Perilipin-1 Proteins 0.000 description 1
- 102000017795 Perilipin-1 Human genes 0.000 description 1
- 102100038883 Peroxisomal biogenesis factor 19 Human genes 0.000 description 1
- 229920005439 Perspex® Polymers 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000235062 Pichia membranifaciens Species 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 102100035194 Placenta growth factor Human genes 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 102100040681 Platelet-derived growth factor C Human genes 0.000 description 1
- 102100040682 Platelet-derived growth factor D Human genes 0.000 description 1
- 101710170209 Platelet-derived growth factor D Proteins 0.000 description 1
- 102100037596 Platelet-derived growth factor subunit A Human genes 0.000 description 1
- 101710103506 Platelet-derived growth factor subunit A Proteins 0.000 description 1
- 102100040990 Platelet-derived growth factor subunit B Human genes 0.000 description 1
- 101710103494 Platelet-derived growth factor subunit B Proteins 0.000 description 1
- 229920000805 Polyaspartic acid Polymers 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 102100038255 Prefoldin subunit 1 Human genes 0.000 description 1
- 102100038619 Prenylated Rab acceptor protein 1 Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- 102000003946 Prolactin Human genes 0.000 description 1
- 101710119112 Prolactin-2 Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102000017975 Protein C Human genes 0.000 description 1
- 102100039063 Protein IL-40 Human genes 0.000 description 1
- 101710200420 Protein IL-40 Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 108010066124 Protein S Proteins 0.000 description 1
- 102000029301 Protein S Human genes 0.000 description 1
- 102100036894 Protein patched homolog 2 Human genes 0.000 description 1
- 101710161395 Protein patched homolog 2 Proteins 0.000 description 1
- 102100023159 Protein sel-1 homolog 1 Human genes 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 101100408135 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) phnA gene Proteins 0.000 description 1
- 102100032319 Putative activator of 90 kDa heat shock protein ATPase homolog 2 Human genes 0.000 description 1
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 description 1
- 108010025832 RANK Ligand Proteins 0.000 description 1
- 102100022491 RNA-binding protein NOB1 Human genes 0.000 description 1
- 102100023433 RNA-binding protein RO60 Human genes 0.000 description 1
- 102100022881 Rab proteins geranylgeranyltransferase component A 1 Human genes 0.000 description 1
- 102100022880 Rab proteins geranylgeranyltransferase component A 2 Human genes 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 108010047909 Resistin Proteins 0.000 description 1
- 102000007156 Resistin Human genes 0.000 description 1
- 102100029826 Reticulon-4 receptor Human genes 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 229940124859 Rotavirus vaccine Drugs 0.000 description 1
- IGLNJRXAVVLDKE-OIOBTWANSA-N Rubidium-82 Chemical compound [82Rb] IGLNJRXAVVLDKE-OIOBTWANSA-N 0.000 description 1
- 108700031620 S-acetylthiorphan Proteins 0.000 description 1
- 108010005173 SERPIN-B5 Proteins 0.000 description 1
- 102100022330 SPRY domain-containing SOCS box protein 2 Human genes 0.000 description 1
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 1
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 1
- 101100323459 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) APJ1 gene Proteins 0.000 description 1
- 101100441892 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CPR7 gene Proteins 0.000 description 1
- 101100125012 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ECM10 gene Proteins 0.000 description 1
- 101100280133 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) EUG1 gene Proteins 0.000 description 1
- 101100012952 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FPR2 gene Proteins 0.000 description 1
- 101100285707 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HSC82 gene Proteins 0.000 description 1
- 101100071588 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HSP12 gene Proteins 0.000 description 1
- 101100071604 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HSP42 gene Proteins 0.000 description 1
- 101100453261 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) JEM1 gene Proteins 0.000 description 1
- 101100236846 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MDJ2 gene Proteins 0.000 description 1
- 101100420794 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SCJ1 gene Proteins 0.000 description 1
- 101100502339 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SFA1 gene Proteins 0.000 description 1
- 101100451671 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SSA3 gene Proteins 0.000 description 1
- 101100451681 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SSA4 gene Proteins 0.000 description 1
- 101100285899 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SSE2 gene Proteins 0.000 description 1
- 101001059240 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Site-specific recombinase Flp Proteins 0.000 description 1
- 101100537260 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) TIM10 gene Proteins 0.000 description 1
- 101100319895 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) YAP3 gene Proteins 0.000 description 1
- 101100160515 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) YPS1 gene Proteins 0.000 description 1
- 241001123227 Saccharomyces pastorianus Species 0.000 description 1
- 108010084592 Saporins Proteins 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 241000242678 Schistosoma Species 0.000 description 1
- 241000242680 Schistosoma mansoni Species 0.000 description 1
- BUGBHKTXTAQXES-AHCXROLUSA-N Selenium-75 Chemical compound [75Se] BUGBHKTXTAQXES-AHCXROLUSA-N 0.000 description 1
- 102000013008 Semaphorin-3A Human genes 0.000 description 1
- 108010090319 Semaphorin-3A Proteins 0.000 description 1
- 102100030333 Serpin B5 Human genes 0.000 description 1
- 101150108455 Sil1 gene Proteins 0.000 description 1
- 241000269456 Silurana Species 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102100036422 Speckle-type POZ protein Human genes 0.000 description 1
- 108010088160 Staphylococcal Protein A Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108700011201 Streptococcus IgG Fc-binding Proteins 0.000 description 1
- 101000880156 Streptomyces cacaoi Subtilisin inhibitor-like protein 1 Proteins 0.000 description 1
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 1
- 102400000096 Substance P Human genes 0.000 description 1
- 101800003906 Substance P Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101000930463 Sus scrofa Albumin Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 102100028679 T-complex protein 1 subunit beta Human genes 0.000 description 1
- 102100029958 T-complex protein 1 subunit delta Human genes 0.000 description 1
- 102100029886 T-complex protein 1 subunit epsilon Human genes 0.000 description 1
- 102100036476 T-complex protein 1 subunit eta Human genes 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- 102100026311 T-complex protein 1 subunit theta Human genes 0.000 description 1
- 102100030664 T-complex protein 1 subunit zeta Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101150001810 TEAD1 gene Proteins 0.000 description 1
- 101150074253 TEF1 gene Proteins 0.000 description 1
- 102000003627 TRPC1 Human genes 0.000 description 1
- 102000003141 Tachykinin Human genes 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 102100031279 Tetratricopeptide repeat protein 4 Human genes 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010061174 Thyrotropin Proteins 0.000 description 1
- 102000011923 Thyrotropin Human genes 0.000 description 1
- 102100030951 Tissue factor pathway inhibitor Human genes 0.000 description 1
- 101710139626 Tissue factor pathway inhibitor Proteins 0.000 description 1
- IWEQQRMGNVVKQW-OQKDUQJOSA-N Toremifene citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 IWEQQRMGNVVKQW-OQKDUQJOSA-N 0.000 description 1
- 101710119887 Trans-acting factor B Proteins 0.000 description 1
- 101710119961 Trans-acting factor C Proteins 0.000 description 1
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000009298 Trigla lyra Species 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 102000013534 Troponin C Human genes 0.000 description 1
- 102000004987 Troponin T Human genes 0.000 description 1
- 108090001108 Troponin T Proteins 0.000 description 1
- 101100472152 Trypanosoma brucei brucei (strain 927/4 GUTat10.1) REL1 gene Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100024568 Tumor necrosis factor ligand superfamily member 11 Human genes 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 1
- 102100033732 Tumor necrosis factor receptor superfamily member 1A Human genes 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 102100035284 Tumor necrosis factor receptor superfamily member 6B Human genes 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 1
- 102100030810 Tumor necrosis factor receptor superfamily member EDAR Human genes 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- 101150050575 URA3 gene Proteins 0.000 description 1
- 101150044776 URA5 gene Proteins 0.000 description 1
- 102400000757 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102100020712 Ubiquitin-conjugating enzyme E2 G1 Human genes 0.000 description 1
- 101710101614 Ubiquitin-conjugating enzyme E2 G1 Proteins 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 108010073919 Vascular Endothelial Growth Factor D Proteins 0.000 description 1
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 1
- 102000016663 Vascular Endothelial Growth Factor Receptor-3 Human genes 0.000 description 1
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- FHNFHKCVQCLJFQ-NJFSPNSNSA-N Xenon-133 Chemical compound [133Xe] FHNFHKCVQCLJFQ-NJFSPNSNSA-N 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- 101001060278 Xenopus laevis Fibroblast growth factor 3 Proteins 0.000 description 1
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 1
- 102100023250 Zinc finger and BTB domain-containing protein 7C Human genes 0.000 description 1
- FPVRUILUEYSIMD-RPRRAYFGSA-N [(8s,9r,10s,11s,13s,14s,16r,17r)-9-fluoro-11-hydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(OC(C)=O)[C@@]1(C)C[C@@H]2O FPVRUILUEYSIMD-RPRRAYFGSA-N 0.000 description 1
- VRWRNFAUYWXDKX-UHFFFAOYSA-N [3-(2,5-dioxopyrrol-1-yl)phenyl]boronic acid Chemical compound OB(O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 VRWRNFAUYWXDKX-UHFFFAOYSA-N 0.000 description 1
- 229940028652 abraxane Drugs 0.000 description 1
- KRHYYFGTRYWZRS-BJUDXGSMSA-N ac1l2y5h Chemical compound [18FH] KRHYYFGTRYWZRS-BJUDXGSMSA-N 0.000 description 1
- XQEJFZYLWPSJOV-XJQYZYIXSA-N acetic acid;(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-pentazacycloicosa Chemical compound CC(O)=O.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 XQEJFZYLWPSJOV-XJQYZYIXSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 229940064305 adrucil Drugs 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229940060238 agrylin Drugs 0.000 description 1
- 229940060236 ala-cort Drugs 0.000 description 1
- 229960005310 aldesleukin Drugs 0.000 description 1
- 229960000548 alemtuzumab Drugs 0.000 description 1
- 229940110282 alimta Drugs 0.000 description 1
- 229960001445 alitretinoin Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229940098174 alkeran Drugs 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 1
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 1
- 238000010976 amide bond formation reaction Methods 0.000 description 1
- 229960001097 amifostine Drugs 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229960001694 anagrelide Drugs 0.000 description 1
- 229960002932 anastrozole Drugs 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 229940115115 aranesp Drugs 0.000 description 1
- 101150042295 arfA gene Proteins 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 229940078010 arimidex Drugs 0.000 description 1
- 229940087620 aromasin Drugs 0.000 description 1
- 229940014583 arranon Drugs 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 1
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 229960002938 bexarotene Drugs 0.000 description 1
- 229960000997 bicalutamide Drugs 0.000 description 1
- 229940108502 bicnu Drugs 0.000 description 1
- 239000012867 bioactive agent Substances 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 229940112133 busulfex Drugs 0.000 description 1
- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical compound CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- KVUAALJSMIVURS-ZEDZUCNESA-L calcium folinate Chemical compound [Ca+2].C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 KVUAALJSMIVURS-ZEDZUCNESA-L 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 229940088954 camptosar Drugs 0.000 description 1
- 229940022399 cancer vaccine Drugs 0.000 description 1
- 238000009566 cancer vaccine Methods 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- OKTJSMMVPCPJKN-BJUDXGSMSA-N carbon-11 Chemical compound [11C] OKTJSMMVPCPJKN-BJUDXGSMSA-N 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 229940097647 casodex Drugs 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000002230 centromere Anatomy 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- 229960005004 cholera vaccine Drugs 0.000 description 1
- 229940048961 cholinesterase Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 229960002303 citric acid monohydrate Drugs 0.000 description 1
- 229960002436 cladribine Drugs 0.000 description 1
- GUTLYIVDDKVIGB-YPZZEJLDSA-N cobalt-57 Chemical compound [57Co] GUTLYIVDDKVIGB-YPZZEJLDSA-N 0.000 description 1
- GUTLYIVDDKVIGB-BJUDXGSMSA-N cobalt-58 Chemical compound [58Co] GUTLYIVDDKVIGB-BJUDXGSMSA-N 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- ALEXXDVDDISNDU-JZYPGELDSA-N cortisol 21-acetate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)C[C@@H]2O ALEXXDVDDISNDU-JZYPGELDSA-N 0.000 description 1
- 229960004544 cortisone Drugs 0.000 description 1
- 229940088547 cosmegen Drugs 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 108060002021 cyanovirin N Proteins 0.000 description 1
- PNZXMIKHJXIPEK-UHFFFAOYSA-N cyclohexanecarboxamide Chemical compound NC(=O)C1CCCCC1 PNZXMIKHJXIPEK-UHFFFAOYSA-N 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229940059359 dacogen Drugs 0.000 description 1
- 229960005029 darbepoetin alfa Drugs 0.000 description 1
- 229960002448 dasatinib Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 229960003109 daunorubicin hydrochloride Drugs 0.000 description 1
- 229940041983 daunorubicin liposomal Drugs 0.000 description 1
- 229940107841 daunoxome Drugs 0.000 description 1
- 229940026692 decadron Drugs 0.000 description 1
- 229960003603 decitabine Drugs 0.000 description 1
- 229940027008 deltasone Drugs 0.000 description 1
- 229960002923 denileukin diftitox Drugs 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 229960003957 dexamethasone Drugs 0.000 description 1
- 229960003657 dexamethasone acetate Drugs 0.000 description 1
- 229960002344 dexamethasone sodium phosphate Drugs 0.000 description 1
- PLCQGRYPOISRTQ-FCJDYXGNSA-L dexamethasone sodium phosphate Chemical compound [Na+].[Na+].C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)COP([O-])([O-])=O)(O)[C@@]1(C)C[C@@H]2O PLCQGRYPOISRTQ-FCJDYXGNSA-L 0.000 description 1
- 229940087410 dexasone Drugs 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 108010057167 dimethylaniline monooxygenase (N-oxide forming) Proteins 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 229940115080 doxil Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- GVGYEFKIHJTNQZ-RFQIPJPRSA-N ecgonine benzoate Chemical compound O([C@@H]1[C@@H]([C@H]2CC[C@@H](C1)N2C)C(O)=O)C(=O)C1=CC=CC=C1 GVGYEFKIHJTNQZ-RFQIPJPRSA-N 0.000 description 1
- 229940099302 efudex Drugs 0.000 description 1
- 239000012039 electrophile Substances 0.000 description 1
- 229940073038 elspar Drugs 0.000 description 1
- 229940000733 emcyt Drugs 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 210000000222 eosinocyte Anatomy 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 229960003388 epoetin alfa Drugs 0.000 description 1
- UYAHIZSMUZPPFV-NJFSPNSNSA-N erbium-169 Chemical compound [169Er] UYAHIZSMUZPPFV-NJFSPNSNSA-N 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- YOMFVLRTMZWACQ-UHFFFAOYSA-N ethyltrimethylammonium Chemical compound CC[N+](C)(C)C YOMFVLRTMZWACQ-UHFFFAOYSA-N 0.000 description 1
- 229940098617 ethyol Drugs 0.000 description 1
- 229940085363 evista Drugs 0.000 description 1
- 238000011066 ex-situ storage Methods 0.000 description 1
- 229960000255 exemestane Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229940012413 factor vii Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 229940012426 factor x Drugs 0.000 description 1
- 229940012444 factor xiii Drugs 0.000 description 1
- 229940043168 fareston Drugs 0.000 description 1
- 229940087861 faslodex Drugs 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229940087476 femara Drugs 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 108090000370 fibroblast growth factor 18 Proteins 0.000 description 1
- 102000003977 fibroblast growth factor 18 Human genes 0.000 description 1
- 229960004177 filgrastim Drugs 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- 108700014844 flt3 ligand Proteins 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- 229960005304 fludarabine phosphate Drugs 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 229940064300 fluoroplex Drugs 0.000 description 1
- 229960001751 fluoxymesterone Drugs 0.000 description 1
- 229960002074 flutamide Drugs 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 229960002258 fulvestrant Drugs 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 108010090623 galactose binding protein Proteins 0.000 description 1
- 102000021529 galactose binding proteins Human genes 0.000 description 1
- 229940006110 gallium-67 Drugs 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229940020967 gemzar Drugs 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 229940084910 gliadel Drugs 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
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- PCHJSUWPFVWCPO-OUBTZVSYSA-N gold-198 Chemical compound [198Au] PCHJSUWPFVWCPO-OUBTZVSYSA-N 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 229960002913 goserelin Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229940083461 halotestin Drugs 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 229940003183 hexalen Drugs 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229940006607 hirudin Drugs 0.000 description 1
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 101150026546 hsa gene Proteins 0.000 description 1
- 101150072994 hsp30 gene Proteins 0.000 description 1
- 102000043282 human CCL1 Human genes 0.000 description 1
- 102000043711 human CCL21 Human genes 0.000 description 1
- 102000043802 human CCL22 Human genes 0.000 description 1
- 102000046157 human CSF2 Human genes 0.000 description 1
- 102000055297 human CXCL1 Human genes 0.000 description 1
- 102000046353 human CXCL2 Human genes 0.000 description 1
- 102000052624 human CXCL8 Human genes 0.000 description 1
- 102000049885 human IL11 Human genes 0.000 description 1
- 102000043448 human IL17RA Human genes 0.000 description 1
- 102000055281 human IL1R2 Human genes 0.000 description 1
- 102000046828 human IL1RAP Human genes 0.000 description 1
- 102000046824 human IL1RN Human genes 0.000 description 1
- 102000054663 human IL4R Human genes 0.000 description 1
- 102000055228 human IL5 Human genes 0.000 description 1
- 102000052611 human IL6 Human genes 0.000 description 1
- 102000050109 human Il16 Human genes 0.000 description 1
- 102000043283 human SERPINE1 Human genes 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 229940116886 human interleukin-6 Drugs 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 229940088013 hycamtin Drugs 0.000 description 1
- 229940096120 hydrea Drugs 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 229950000785 hydrocortisone phosphate Drugs 0.000 description 1
- 229960004204 hydrocortisone sodium phosphate Drugs 0.000 description 1
- 229960001401 hydrocortisone sodium succinate Drugs 0.000 description 1
- VWQWXZAWFPZJDA-CGVGKPPMSA-N hydrocortisone succinate Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)COC(=O)CCC(O)=O)[C@@H]4[C@@H]3CCC2=C1 VWQWXZAWFPZJDA-CGVGKPPMSA-N 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 229940099279 idamycin Drugs 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229940090411 ifex Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- 101150045896 ilv-2 gene Proteins 0.000 description 1
- 229960002411 imatinib Drugs 0.000 description 1
- 229960003685 imatinib mesylate Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 229940055742 indium-111 Drugs 0.000 description 1
- APFVFJFRJDLVQX-AHCXROLUSA-N indium-111 Chemical compound [111In] APFVFJFRJDLVQX-AHCXROLUSA-N 0.000 description 1
- 229960003971 influenza vaccine Drugs 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229950000038 interferon alfa Drugs 0.000 description 1
- 229960003521 interferon alfa-2a Drugs 0.000 description 1
- 102000002467 interleukin receptors Human genes 0.000 description 1
- 108010093036 interleukin receptors Proteins 0.000 description 1
- 229940074383 interleukin-11 Drugs 0.000 description 1
- 229940076264 interleukin-3 Drugs 0.000 description 1
- 108010038415 interleukin-8 receptors Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229940065638 intron a Drugs 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- XMBWDFGMSWQBCA-YPZZEJLDSA-N iodane Chemical compound [125IH] XMBWDFGMSWQBCA-YPZZEJLDSA-N 0.000 description 1
- 229940044173 iodine-125 Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- XQZXYNRDCRIARQ-LURJTMIESA-N iopamidol Chemical compound C[C@H](O)C(=O)NC1=C(I)C(C(=O)NC(CO)CO)=C(I)C(C(=O)NC(CO)CO)=C1I XQZXYNRDCRIARQ-LURJTMIESA-N 0.000 description 1
- 229960004647 iopamidol Drugs 0.000 description 1
- 229940084651 iressa Drugs 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- DNNSSWSSYDEUBZ-OIOBTWANSA-N krypton (81mKr) gas Chemical compound [81Kr] DNNSSWSSYDEUBZ-OIOBTWANSA-N 0.000 description 1
- 229960001299 krypton (81mkr) gas Drugs 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 229960004942 lenalidomide Drugs 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229960003881 letrozole Drugs 0.000 description 1
- 229940063725 leukeran Drugs 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 239000013541 low molecular weight contaminant Substances 0.000 description 1
- 229940087857 lupron Drugs 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- 108010019677 lymphotactin Proteins 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 229940087732 matulane Drugs 0.000 description 1
- 229940087412 maxidex Drugs 0.000 description 1
- 101150098728 mdj1 gene Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960002868 mechlorethamine hydrochloride Drugs 0.000 description 1
- QZIQJVCYUQZDIR-UHFFFAOYSA-N mechlorethamine hydrochloride Chemical compound Cl.ClCCN(C)CCCl QZIQJVCYUQZDIR-UHFFFAOYSA-N 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 229940064748 medrol Drugs 0.000 description 1
- 229940090004 megace Drugs 0.000 description 1
- 229960001786 megestrol Drugs 0.000 description 1
- 229960004296 megestrol acetate Drugs 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229960004635 mesna Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000006109 methionine Nutrition 0.000 description 1
- BKBBTCORRZMASO-ZOWNYOTGSA-M methotrexate monosodium Chemical compound [Na+].C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C([O-])=O)C=C1 BKBBTCORRZMASO-ZOWNYOTGSA-M 0.000 description 1
- 229960003058 methotrexate sodium Drugs 0.000 description 1
- 101150072699 mge1 gene Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 229940087004 mustargen Drugs 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229940090009 myleran Drugs 0.000 description 1
- UATCLPJEZJKNHE-UHFFFAOYSA-N n-(3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-yl)-2-iodoacetamide Chemical compound O1C(=O)C2=CC(NC(=O)CI)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 UATCLPJEZJKNHE-UHFFFAOYSA-N 0.000 description 1
- LBWFXVZLPYTWQI-IPOVEDGCSA-N n-[2-(diethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C LBWFXVZLPYTWQI-IPOVEDGCSA-N 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 239000000692 natriuretic peptide Substances 0.000 description 1
- 229940086322 navelbine Drugs 0.000 description 1
- 229960000801 nelarabine Drugs 0.000 description 1
- 229940082926 neumega Drugs 0.000 description 1
- 229940029345 neupogen Drugs 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 229940032018 neurotrophin 3 Drugs 0.000 description 1
- 229940097998 neurotrophin 4 Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229940080607 nexavar Drugs 0.000 description 1
- 229940099637 nilandron Drugs 0.000 description 1
- 229940109551 nipent Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229960002700 octreotide Drugs 0.000 description 1
- 229960001494 octreotide acetate Drugs 0.000 description 1
- 101150087557 omcB gene Proteins 0.000 description 1
- 101150115693 ompA gene Proteins 0.000 description 1
- 101150093139 ompT gene Proteins 0.000 description 1
- 229940100027 ontak Drugs 0.000 description 1
- 108010046821 oprelvekin Proteins 0.000 description 1
- 229940003515 orapred Drugs 0.000 description 1
- 229960001756 oxaliplatin Drugs 0.000 description 1
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- QVGXLLKOCUKJST-BJUDXGSMSA-N oxygen-15 atom Chemical compound [15O] QVGXLLKOCUKJST-BJUDXGSMSA-N 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 101800000857 p40 protein Proteins 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229960002502 paclitaxel protein-bound Drugs 0.000 description 1
- 229940046231 pamidronate Drugs 0.000 description 1
- 229960001972 panitumumab Drugs 0.000 description 1
- 229940096763 panretin Drugs 0.000 description 1
- 229960002566 papillomavirus vaccine Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 229940097097 pediapred Drugs 0.000 description 1
- HQQSBEDKMRHYME-UHFFFAOYSA-N pefloxacin mesylate Chemical compound [H+].CS([O-])(=O)=O.C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCN(C)CC1 HQQSBEDKMRHYME-UHFFFAOYSA-N 0.000 description 1
- 229960001744 pegaspargase Drugs 0.000 description 1
- 108010001564 pegaspargase Proteins 0.000 description 1
- 108010044644 pegfilgrastim Proteins 0.000 description 1
- 229960001373 pegfilgrastim Drugs 0.000 description 1
- 101150040383 pel2 gene Proteins 0.000 description 1
- 101150050446 pelB gene Proteins 0.000 description 1
- 229960005079 pemetrexed Drugs 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- 108010037251 peptide T1249 Proteins 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- JTJMJGYZQZDUJJ-UHFFFAOYSA-N phencyclidine Chemical compound C1CCCCN1C1(C=2C=CC=CC=2)CCCCC1 JTJMJGYZQZDUJJ-UHFFFAOYSA-N 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 229960005141 piperazine Drugs 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108010017992 platelet-derived growth factor C Proteins 0.000 description 1
- 229940063179 platinol Drugs 0.000 description 1
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 1
- 229920000889 poly(m-phenylene isophthalamide) Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 108010011110 polyarginine Proteins 0.000 description 1
- 108010064470 polyaspartate Proteins 0.000 description 1
- 108010077051 polycysteine Proteins 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 229940096111 prelone Drugs 0.000 description 1
- 108010071286 prethrombins Proteins 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 229940029359 procrit Drugs 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 229940087463 proleukin Drugs 0.000 description 1
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 1
- 229940080818 propionamide Drugs 0.000 description 1
- 108010076038 prosaptide Proteins 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000018883 protein targeting Effects 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 229940117820 purinethol Drugs 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 229960004622 raloxifene Drugs 0.000 description 1
- 108091007054 readthrough proteins Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 229940120975 revlimid Drugs 0.000 description 1
- WUAPFZMCVAUBPE-IGMARMGPSA-N rhenium-186 Chemical compound [186Re] WUAPFZMCVAUBPE-IGMARMGPSA-N 0.000 description 1
- 229940061969 rheumatrex Drugs 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 102200091899 rs75353611 Human genes 0.000 description 1
- 102220105280 rs879254406 Human genes 0.000 description 1
- 108091008601 sVEGFR Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- KZUNJOHGWZRPMI-AKLPVKDBSA-N samarium-153 Chemical compound [153Sm] KZUNJOHGWZRPMI-AKLPVKDBSA-N 0.000 description 1
- 229940072272 sandostatin Drugs 0.000 description 1
- 108700014314 sandostatinLAR Proteins 0.000 description 1
- 108010038379 sargramostim Proteins 0.000 description 1
- 229960002530 sargramostim Drugs 0.000 description 1
- 108010073863 saruplase Proteins 0.000 description 1
- 238000013077 scoring method Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 101150017313 sls1 gene Proteins 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000012321 sodium triacetoxyborohydride Substances 0.000 description 1
- MKNJJMHQBYVHRS-UHFFFAOYSA-M sodium;1-[11-(2,5-dioxopyrrol-1-yl)undecanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCCCCCCN1C(=O)C=CC1=O MKNJJMHQBYVHRS-UHFFFAOYSA-M 0.000 description 1
- ULARYIUTHAWJMU-UHFFFAOYSA-M sodium;1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O ULARYIUTHAWJMU-UHFFFAOYSA-M 0.000 description 1
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 1
- HHSGWIABCIVPJT-UHFFFAOYSA-M sodium;1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 HHSGWIABCIVPJT-UHFFFAOYSA-M 0.000 description 1
- MIDXXTLMKGZDPV-UHFFFAOYSA-M sodium;1-[6-(2,5-dioxopyrrol-1-yl)hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O MIDXXTLMKGZDPV-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 229940088542 solu-cortef Drugs 0.000 description 1
- 229940087854 solu-medrol Drugs 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 229960003787 sorafenib Drugs 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 108010028621 stem cell inhibitory factor Proteins 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 229940006509 strontium-89 Drugs 0.000 description 1
- CIOAGBVUUVVLOB-OUBTZVSYSA-N strontium-89 Chemical compound [89Sr] CIOAGBVUUVVLOB-OUBTZVSYSA-N 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960001796 sunitinib Drugs 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940034785 sutent Drugs 0.000 description 1
- 229940095374 tabloid Drugs 0.000 description 1
- 108060008037 tachykinin Proteins 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 229940120982 tarceva Drugs 0.000 description 1
- 229940099419 targretin Drugs 0.000 description 1
- RCINICONZNJXQF-XAZOAEDWSA-N taxol® Chemical compound O([C@@H]1[C@@]2(CC(C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3(C21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-XAZOAEDWSA-N 0.000 description 1
- 229940063683 taxotere Drugs 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- 229940056501 technetium 99m Drugs 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- 229940061353 temodar Drugs 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- BKVIYDNLLOSFOA-OIOBTWANSA-N thallium-201 Chemical compound [201Tl] BKVIYDNLLOSFOA-OIOBTWANSA-N 0.000 description 1
- 229940034915 thalomid Drugs 0.000 description 1
- 229940110675 theracys Drugs 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000034005 thiol-disulfide exchange Effects 0.000 description 1
- 229960001196 thiotepa Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 108010065972 tick anticoagulant peptide Proteins 0.000 description 1
- 210000001578 tight junction Anatomy 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 229940035307 toposar Drugs 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- 229960005026 toremifene Drugs 0.000 description 1
- XFCLJVABOIYOMF-QPLCGJKRSA-N toremifene Chemical compound C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 XFCLJVABOIYOMF-QPLCGJKRSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000013715 transcription antitermination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000012863 translational readthrough Effects 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940086984 trisenox Drugs 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 101150044170 trpE gene Proteins 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229940094060 tykerb Drugs 0.000 description 1
- 101150070177 ubi4 gene Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 229940005267 urate oxidase Drugs 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
- 229940099039 velcade Drugs 0.000 description 1
- 229940065658 vidaza Drugs 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- 229960004982 vinblastine sulfate Drugs 0.000 description 1
- AQTQHPDCURKLKT-JKDPCDLQSA-N vincristine sulfate Chemical compound OS(O)(=O)=O.C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C=O)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 AQTQHPDCURKLKT-JKDPCDLQSA-N 0.000 description 1
- GBABOYUKABKIAF-IELIFDKJSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-IELIFDKJSA-N 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 description 1
- 229960002166 vinorelbine tartrate Drugs 0.000 description 1
- GBABOYUKABKIAF-IWWDSPBFSA-N vinorelbinetartrate Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC(C23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-IWWDSPBFSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 229960001134 von willebrand factor Drugs 0.000 description 1
- 229960000237 vorinostat Drugs 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
- 229940053867 xeloda Drugs 0.000 description 1
- 229960003267 xenon (127xe) gas Drugs 0.000 description 1
- FHNFHKCVQCLJFQ-AHCXROLUSA-N xenon-127 Chemical compound [127Xe] FHNFHKCVQCLJFQ-AHCXROLUSA-N 0.000 description 1
- 229940106670 xenon-133 Drugs 0.000 description 1
- 229940053890 zanosar Drugs 0.000 description 1
- 229940033942 zoladex Drugs 0.000 description 1
- 229960004276 zoledronic acid Drugs 0.000 description 1
- 229940061261 zolinza Drugs 0.000 description 1
- 229940002005 zometa Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
-
- G06F19/12—
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to conjugation competent albumins and albumin-related polypeptides, and their conjugates with at least one moiety, and to polynucleotides encoding them.
- Serum albumins provide valuable scaffolds to which bioactive molecules may be fused, either through genetic fusions or chemical fusions to improve the properties of the fused molecule(s) (Leger, R. et al. (2004). Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau, K., et al. (2005). Bioconjug Chem 16(4): 1000-8; Balan, V. et al. (2006). Antivir Ther 11(1): 35-45; EP 0 413 622; WO 90/13653; EP 1 681 304; WO 1997/024445; WO 01/79271).
- Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused bioactive molecule from inactivation.
- HSA Human serum albumin
- SEQ ID No. 1 The sequence of HSA is provided in SEQ ID No. 1. Natural variants of HSA occur and a list of know polymorphisms is given in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and at http://www.uniprot.org/uniprot/P02768.
- the HSA polypeptide chain has 35 cysteine residues, which form 17 disulphide bonds and one unpaired (free) cysteine at position 34 of the mature protein (Seq ID No. 1).
- Cysteine-34 has been used to for conjugation of molecules to albumin (Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8), and provides a precise, well defined site for conjugation.
- conjugation at cysteine-34 provides only one site for attachment of a single moiety thus there is no choice of conjugation site.
- the provision of a single conjugation sites means that only one moiety can be conjugated to each albumin molecule. What is required is an albumin molecule which provides one or more alternative attachment sites.
- thio-albumin is used herein to describe an albumin variant which comprises one or more unpaired cysteine residues, particularly an albumin variant in which one or more of the unpaired cysteine residues does not occur in a naturally occurring variant of an albumin.
- a thio-albumin is a ‘conjugation competent albumin’.
- a thio-albumin may be referred to as a ‘cysteine variant of an albumin’.
- albumin includes naturally occurring albumin, albumin-related proteins and variants thereof such as natural and engineered variants. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins.
- the albumin may have at least 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99% similarity or identity to SEQ ID No. 1.
- a thio-albumin of the invention may be a derivative of, or be based on, any of such albumin.
- the unpaired cysteine residues may be provided by insertion, deletion, substitution, addition or extension of an albumin sequence.
- the invention also relates to a conjugate comprising at least one, for example 2, 3, 4, 5 or 6, conjugation partners such as bioactive compounds and a polypeptide according to the invention
- the invention also provides a method for designing conjugation-competent albumins.
- FIG. 1 is a table showing criteria used to select sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of conjugation competent cysteines.
- the protein sequences include the leader sequence.
- the protein sequences include the leader sequence.
- FIG. 4 is a Venn diagram showing the classes of and relationship between twenty amino acids.
- FIGS. 5A, 5B, 5C and 5D are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of one or more conjugation competent cysteine.
- FIGS. 6A and 6B are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for disruption of one or more disulphide bonds for the generation of one or more conjugation competent cysteines.
- FIG. 7 is a map of plasmid pDB2244.
- FIG. 8 is a map of plasmid pDB2243.
- FIG. 9 is a map of plasmid pDB2713.
- FIG. 10 is a table showing preferred sites for conjugation grouped according to their relative position on a folded albumin of SEQ ID No. 1.
- FIG. 11 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having a single free thiol group in addition to Cys-34 of native human serum albumin.
- FIG. 12 is a map of plasmid pDB3927.
- FIG. 13 is a map of plasmid pDB3964.
- FIG. 14 is a map of plasmid pBD3936.
- FIG. 15 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation) of albumin molecules having a free thiol group at Cys-34 of SEQ ID No. 1 and an additional free-thiol at the position indicated below the bar chart.
- FIG. 16 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed.
- FIG. 17 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation of albumin molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed.
- FIG. 18 is a table showing the fermentation yield and relative level of conjugation to albumin molecules comprising one or more free-thiols.
- FIG. 19 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) before treatment with DTNB.
- FIG. 20 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) after treatment with DTNB.
- FIG. 21 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) before treatment with DTNB.
- FIG. 22 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) after treatment with DTNB.
- FIG. 23 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol) before treatment with DTNB.
- FIG. 24 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol).
- a first aspect of the invention provides a method for designing and/or preparing variant albumins comprising one or more conjugation competent cysteine residues. Therefore, the polypeptide may be considered to be conjugation-competent.
- an albumin may be referred to as a ‘thio-albumin’ or as a ‘cysteine varant’ of an albumin.
- conjugation competent cysteine includes a cysteine which has a thiol which is not disulphide bonded to another cysteine and which is, preferably, not blocked from conjugating to another molecule (which may be referred to as a ‘conjugation partner’) due to unfavourable steric hindrances. That is, preferably the location of the cysteine within or on a folded polypeptide is such that it is available for conjugation.
- a number of selection criteria may or may not be used alone or in any combination in order to identify suitable sites for introduction of a conjugation competent cysteine residue. Therefore, the invention provides a method and/or rules for a priori identification of sites of an amino acid sequence of albumin at which a conjugation competent cysteine may be introduced. Such sites may be referred to as ‘candidate residues’.
- the albumin sequence on which the variant albumin is based may be SEQ ID No. 1 or any other albumin.
- the variant albumin may be be based on an albumin which does or does not have a cysteine at position 34 of the amino acid sequence, or an equivalent position.
- Cysteine residues may or may not be introduced by one or more of substitution, insertion, deletion, extension and addition. Sites may or may not be selected with reference to a 3-dimensional structure of an albumin or variant thereof. The following criteria may or may not be used to select suitable sites:
- the surface accessibility is high.
- the surface accessibility is at least 60%, more preferably, from 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% to 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%.
- % SASA may be determined as a ‘raw score’ using the methods described herein or may be calculated relative to the score of the residue which has the maximum surface accessibility in the protein.
- the albumin of HSA 1AO6 has a maximum surface accessibility of 229.0 and this is the highest scoring residue in HSA.
- a higher surface accessibility indicates that the residue is on the surface of the protein and is therefore available for binding. Such accessibility may be calculated using a method as described herein.
- the B-factor indicates relative flexibility of an amino acid residue within a 3-dimensional structure.
- the B-factor is from at least 30, 40, 50, 60, 70, 80 or 90% to at least 40, 50, 60, 70, 80, 90 or 100% which may or may not be relative to the maximal B-factor score of any amino acid residue within the molecule.
- HSA e.g. 1AO6
- the B-factor score is high, for example from at least 30, 40, 50, 60, 70, 80, 90, or 100 to at least 40, 50, 60, 70, 80, 90, 100 or 106 (for example using the B-factor scoring system described herein).
- the B-factor score may be less than or equal to 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10%, as described herein.
- the B-Factor (root mean square fluctuations) of the C-alpha carbon atoms during the last nanosecond of the simulation may be calculated using the Gromacs tool “g_rmsf”, version 3.3, based on D. van der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark and H. J. C. Berendsen: GROMACS: Fast, Flexible and Free, J. Comp. Chem. 26 pp. 1701-1718 (2005).
- the candidate residue may or may not be located within secondary structure for example H (Helix), B (isolated beta bridge) or E (Extended sheet). Location of the residue outside of secondary structure indicates that the residue is less likely to be important to secondary structure and/or is more likely to be available for binding than a residue located within secondary structure.
- a candidate residue shows a homology of less than 100% relative to alignment of the albumin in which the residue is located with known albumins (e.g. mammalian albumins such as those shown in FIG. 2 or a combination of mammalian and non-mammalian albumins such as those shown in FIG. 3 ).
- albumins e.g. mammalian albumins such as those shown in FIG. 2 or a combination of mammalian and non-mammalian albumins such as those shown in FIG. 3 ).
- a homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 is preferred.
- Homology can be determined using algorithms known in the art such as Clustal, e.g. Clustal W (Thompson et al. (1994). Nucleic Acids Res 22(22): 4673-80) or Clustal V (Higgins, D. G. and P. M. Sharp (1989). “Fast and sensitive multiple sequence alignments on a microcomputer.” Comput Appl Biosci 5(2): 151-3.). Lower homology indicates that the residue is not particularly important or critical to the structure and/or function of the protein. Preferably the homology is determined with reference to the sixteen mammalian albumins of FIG. 2 or the thirty three mammalian and non-mammalian albumins of FIG. 3 .
- each residue has one or two adjacent residues. If a candidate residue is immediately adjacent one or more residues having a low homology, relative to known albumins, this indicates that the candidate residue is unlikely to be particularly important or critical to the structure and/or function of the protein. This is because the candidate residue is likely to be located within a relatively unconserved region of the protein. It is therefore preferred that the candidate residue is not adjacent a residue which has 100% homology relative to alignment of the albumin with known albumins. Homology may be determined as described herein.
- the candidate residue may be adjacent two residues (i.e.
- the candidate residue is adjacent one or two residues having a homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5—these levels of homology may be referred to as ‘thresholds’.
- Homology may be determined as described herein.
- the location of an amino acid relative to a conserved region may be quantified, for example by scoring an amino acid which is not adjacent any amino acid exceeding the homology threshold as 0, scoring an amino acid which is adjacent one amino acid exceeding the threshold as 1 and scoring an amino acid which adjacent two amino acids exceeding the threshold as 2.
- a polymorphism is a genetic variation, a polymorphism may or may not cause a phenotypic change to the resultant protein.
- the candidate residue is not at a position for which a polymorphism causing a phenotypic change is known. More preferably, the candidate residue is not at a position for which a polymorphism causes, or is known to cause, thermal instability.
- Polymorphisms known for HSA SEQ ID No. 1 are detailed in FIG. 1 and are also discussed in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16 and at http://www.uniprot.org/uniprot/P02768.
- the presence, absence and/or effect of a polymorphism may be quantified, for example by scoring a known polymorphism that has no phenotypic change as 0, scoring a polymorphism where a phenotypic change is known (but not known to cause thermal instability) as 1 and scoring a polymorphism which is known to cause thermal instability as 2.
- FIG. 4 is a Venn diagram which provides one system by which conservation level can be quantified.
- the scoring system of FIG. 4 uses a scale of 0 to 5 in which substitutions of high conservation have a score of 0, substitutions of low conservation have a score of 5 and substitutions of intermediate conservation have a score of 1, 2, 3 or 4.
- substitution of the candidate residue is not an unconserved substitution, that is preferably (using the scoring system of FIG. 4 ) the candidate residue does not have conservation score (relative to cysteine) of 5. More preferably the candidate residue has a higher conservation relative to cysteine (e.g. a score of 4, 3, 2 and, more preferably, 1).
- the scoring system is described in the section entitled ‘Conservative Substitution’ (below).
- the thio-albumin may or may not be capable of being expressed at a level of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to the expression of an unmodified albumin (such as SEQ ID No. 1) from a suitable expression system, such as yeast (e.g. Saccharomyces , e.g. S. cerevisiae ) or an Aspergillus .
- yeast e.g. Saccharomyces , e.g. S. cerevisiae
- Aspergillus e.g. Saccharomyces , e.g. S. cerevisiae
- Relative expression levels can be determined, for example, by expression of the protein followed by quantification by SDS-PAGE, HPLC or Western Blotting.
- the thio-albumin may or may not have a high level of conjugation competence, for example at least 50, 60, 70, 80, 90, 95 or 100% relative to the conjugation competence of an albumin consisting of SEQ ID No. 1 having only one conjugation competent cysteine at Cys-34.
- Conjugation competence may be determined relative to any conjugatable molecule (conjugation partner) of interest, for example a bioactive molecule or a fluorescent dye. Determination may be through mass spectrometry analysis or quantification of the activity of the bioactive compound such as its fluorescence.
- An advantage of a thio-albumin having a high conjugation competence is that it may allow efficient conjugation of molecules to the thio-albumin.
- Conjugation competence may be measured with respect to time.
- Favoured thio-albumins may be (a) those which achieve maximal conjugation quickly or (b) slowly.
- the thio-albumin of the invention may be conjugated to a compound (conjugation partner), for example a bioactive compound, such that the compound has a high level of activity relative to its activity in an unconjugated state.
- a conjugated compound shows at least 1, 10, 20, 40, 50, 60, 70, 80, 80 and most preferably 100% of its activity relative to its unconjugated state.
- the conjugated- and/or non-conjugated thio-albumin may or may not have a receptor binding activity of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the receptor binding activity of human serum albumin (SEQ ID No. 1).
- the conjugated- and/or non-conjugated thio-albumin may or may not have a lower receptor binding activity for example at most 0, 10, 20, 30, 40, 50, 60, 70, 80 or 90% than human serum albumin.
- Receptor binding activity may be determined by assay, such as in relation to binding to FcRn.
- FIG. 1 shows the scores of each amino acid residue of HSA (SEQ ID No. 1) for each of parameters (a) to (g).
- HSA is initially produced as a 609 amino acid protein in which the first twenty four amino acids are a leader sequence. The leader sequence is cleaved off to generate a 585 amino acid mature protein. Throughout this specification, the mature protein is referred to as SEQ ID No. 1.
- the structure of HSA model A106 disregards the first four residues and the last three residues of SEQ ID No. 1 because these are unresolved in the 3D model. Therefore, residue 1 of model A106 is equivalent to residue 5 of SEQ ID No. 1. Throughout this specification, all residues are cited with reference to SEQ ID No. 1, unless stated otherwise.
- the immature sequence of HSA i.e. HSA with its natural C-terminal leader sequence
- Position in 1AO6 refers to the amino acid position in the crystal structure of human serum albumin from the RCSD Protein Databank (PDB, http://www.rcsb.org/pdb/) with the entry with PDB identity 1AO6 or 1ao6 (Sugio, S., A. Kashima, et al. (1999). Protein Eng 12(6): 439-46). Note that compared to the mature HSA sequence (SEQ ID No1), the 1AO6 structure starts at residue 5S (with the first 4 amino acids absent from the structure) and finishes at 582A of SEQ ID No1 (with the last 3 amino acids absent from the structure).
- the amino acid positions used herein to describe positions to alter to generate conjugation competent cysteines are referring to the positions in SEQ ID No1, not 1ao6.
- Position in Mature HSA The amino acid position in SEQ ID No. 1 taken from the 585 residue secreted form of HSA, National Center for Biotechnology Information, ACCESSION: 1AO6_A VERSION GI:3212456 (24-SEP.-2008), Chain A, Crystal Structure Of Human Serum Albumin. (Sugio et al. (1999). Protein Eng 12(6): 439-46).
- Position with Leader Sequence refers to the position in the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence.
- % SASA The solvent accessible surface area calculated for each residue, using the DSSP software described in Kabschand and Sander (1983). Biopolymers 22(12): 2577-637. Each solvent accessible surface area was divided by a standard value for the particular amino acid found in that position and multiplied by 100, thereby obtaining a percentage of the standard value for each residue.
- B-Factor The crystallographic B-factor value for the C-alpha atom was extracted directly from the PDB file.
- the B-factor is in column number 11 of the 1ao6 PDB file PDB, (http://www.rcsb.org/pdb/).
- SS Secondary Structure: The secondary structure determined for each residue using the DSSP software Kabsch and Sander (1983). Biopolymers 22(12): 2577-637. If the secondary structure is defined as H (Helix), B (isolated beta bridge) or E (Extended sheet), the residue is marked ‘1’, otherwise as ‘0’.
- Adj. 100%'s (Align 1): The score according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with the mammalian albumins of FIG. 2 .
- a score of 0 indicates the residue is not adjacent to a residue with 100% homology when HSA is aligned with the mammalian albumins of FIG. 2 ;
- a score of 1 indicates that the residue is adjacent to one residue with 100% homology when HSA is aligned with mammalian albumins;
- a score of 2 indicates that the residue is adjacent to two residue with 100% homology when HSA is aligned with mammalian albumins.
- Polymorph This identifies whether or not a polymorphism is known at the amino acid residue.
- Single amino acid polymorphisms of human serum albumin SEQ ID NO: 1 were taken from Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768, with amino acid positions taken from the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence, and described using the standard one letter amino acid code (e.g. D25V refers to an aspartic acid being changed to a valine at position 1 in SEQ ID NO: 1).
- Cysteine A score referring to how well conserved the amino acid is compared to cysteine, as derived from FIG. 4 (described herein), and ranging from 1 to 5 for mutations to cysteine. A score of 1 is assigned to the most conservative changes possible (e.g. alanine to cysteine), and ranging to a score of 5 for the lowest of conservation compared to a cysteine (e.g. histidine to cysteine).
- selection criteria can be used in any desired combination, four preferred groups of selection criteria (A, B, C, D) are described, by way of example only, below. Of these (A) and (B) may also be referred to as Selection Groups 1 and 2 (respectively):
- a particularly preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence which corresponds to the first, second, third, fourth or fifth residue relative to the N- or C-terminus of the albumin sequence (of the model or of the amino acid sequence) or which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility (SASA) of at least 90%; B-factor score of at least 60; less than 80% homology to known mammalian albumins (e.g. FIG. 2 ); no adjacent residues with 100% homology to known mammalian albumins (e.g. FIG. 2 ); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above;
- SASA surface accessibility
- each alteration is an amino acid deletion, substitution, extension, addition or insertion
- candidate residues identified by selection criteria (A) include L585, D1, A2, D562, A364, A504, E505, T79 and E86 (in descending order of solvent accessibility) and are also shown in FIG. 5A .
- Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 90%; B-factor score of at least 40; less than 80% homology to known mammalian albumins (e.g. FIG. 2 ); no adjacent residues with 100% homology to known mammalian albumins (e.g. FIG. 2 ); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above;
- each alteration is an amino acid deletion, substitution, extension, addition or insertion
- candidate residues identified by selection criteria include D129, D549, A581, D121, E82, S270, Q397 and A578 (in descending order of solvent accessibility) and are also shown in FIG. 5B .
- Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 50; less than 100% homology to known mammalian albumins (e.g. FIG. 2 ); less than 80% homology to the various albumins aligned in FIG. 3 ; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above;
- each alteration is an amino acid deletion, substitution, extension, addition or insertion
- candidate residues identified by selection criteria (C) are shown in FIG. 5C .
- the three dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model);
- selecting a candidate amino acid residue in the albumin sequence which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 30; less than 100% homology to known mammalian albumins (e.g. FIG. 2 ); less than 80% homology to the various albumins aligned in FIG. 3 ; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above;
- each alteration is an amino acid deletion, substitution, extension, addition or insertion
- candidate residues identified by selection criteria (D) are shown in FIG. 5D .
- FIGS. 5A, 5B, 5C and 5D are selections from FIG. 1 , the column headings are the same.
- a candidate residue may be one or more of the cysteine residues involved in disulphide bonding present in the albumin molecule (in the case of HSA, SEQ ID No. 1, there are 17 disulphide bonds and therefore 34 cysteines involved in disulphide bonding).
- Two cysteines which are linked by a disulphide bond may be referred to as ‘counterparts’.
- the candidate residue In order to generate a conjugation competent cysteine, the candidate residue may be deleted or may be substituted with a different amino acid, particularly Ser, Thr, Val or Ala in order to create a free thiol at the partner cysteine.
- Cysteine residues were visually inspected using the PyMOL software (Warren L. DeLano “The PyMOL Molecular Graphics System.” DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org), and the cysteines in the disulphide bonds were divided into 3 categories:
- the judgment is based on surface accessibility and the orientation of the C-alpha -C-beta bond of the potential free thiol relative to the folded polypeptide. Using this judgment each of the cysteine residues of HSA were given a modification score and ranked as high, medium or low.
- FIG. 6A provides a list of all the cysteine residues which have a high modification score (right hand column), indicating that modification of a cysteine residue at this position would result in its counterpart cysteine providing a free thiol that has a high probability of being suitable for use as a conjugation site.
- FIG. 6B provides a list of the counterpart cysteines that that, when unpaired (thus providing a free thiol), have a high probability of being suitable for use as a conjugation site
- Modification Score defined as ‘high’, ‘medium’ or low' as described herein.
- Disulphide Information summarises disulphide pairing in SEQ ID No. 1.
- Phenotype Change summarises the columns labelled ‘Polymorph.’ And ‘Phenotype Change’ in FIG. 1 .
- cysteine residues for modification could be further selected based on the other information provided in FIG. 6A , such as assigned secondary structure, cysteine residues with no adjacent conserved residues (100% amongst mammalian albumins (aligned by Clustal W), no known polymorphisms causing phenotypic changes.
- cysteine residues for modification could be selected by examining the environment of the cysteine residue (containing a free thiol group) generated by modification of its counterpart cysteine residue provided in FIG. 6A , characteristics such as high % SASA may be preferred ( FIG. 6B , fifth column).
- the selection criteria of Group (A) are more preferable than those of Group (B) which in turn are more preferable than those of Group (C) and in turn are more preferable than those of Group (D).
- the method may or may not further comprise determining the receptor binding capacity and/or the conjugation competence of the polypeptide and optionally selecting a polypeptide which does or does not have a receptor binding capacity and/or conjugation competence.
- Preparing a polypeptide may or may not include expressing the polypeptide in a host cell and/or purifying the polypeptide from the host or host cell media.
- the method may comprise favouring selection of residues meeting one or all of the following criteria:
- selection criteria as detailed throughout this specification may or may not be used to select residues in the method of the first aspect of the invention.
- a second aspect of the invention provides a thio-albumin comprising a polypeptide sequence and/or polypeptide designed and/or produced according to the first aspect of the invention.
- polypeptide is a recombinant polypeptide.
- polypeptide is an isolated and/or purified polypeptide.
- polypeptide is synthetic and/or does not naturally occur in nature.
- the invention provides a polypeptide which has an amino acid sequence which is at least 60% identical to residues 1 to 585 of SEQ ID No. 1 or a fragment or fusion thereof, in which:
- conjugation competent polypeptide comprising an amino acid sequence which is at least 60% identical to residues 1 to 585 of SEQ ID No. 1, or a fragment or fusion thereof, in which:
- conjugation competent cysteine residues preferably 2 or more or 3 or more.
- the polypeptide may or may not comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conjugation competent cysteine residues.
- polypeptide (which may be described in relation to a known albumin sequence such as SEQ ID No. 1) may or may not comprise one or more of:
- a cysteine with a free thiol group at a position which may or may not correspond to any of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 which may or may not be generated by deletion or substitution of C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567.
- a thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1), is modified to an amino acid which is not cysteine.
- cysteine may or may not be replaced by an amino acid which has a relatively high conservation score (e.g. 1, 2 or 3 as calculated according to FIG. 4 ) such as alanine or serine.
- a thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1) are present.
- the invention may be achieved by introducing cysteine residues by one or more of extension, addition, insertion, substitution or deletion.
- An addition may be made by extension and/or insertion.
- one or more conjugation competent cysteines may or may not be created in an albumin by extension; e.g. by adding an extra cysteine residue to the N-terminus or C-terminus of the molecule, which may or may not be added as a single cysteine residue, or as a longer polypeptide which contains one or more conjugation competent cysteines.
- the cysteine residue(s) may be added immediately adjacent the N- or C-terminus of the albumin.
- cysteine residues When two or more cysteine residues are added, some or all of the added cysteines may be separated from each other by one or more other amino acids, for example by from 1 to 50 amino acids, such as from 1, 10, 20, 30, or 40 amino acids to from 10, 20, 30, 40, or 50 amino acids.
- a preferred N-terminal extension is the addition of Cys immediately adjacent the N-terminal of a mature albumin (i.e. albumin cleaved from its leader sequence).
- Cys is preferably immediately N-terminal to the first Asp (D1).
- Such an albumin may be referred to as ‘Cys-albumin’, e.g. ‘Cys-HSA’ (where HSA is Human Serum Albumin).
- Cys-Ala-albumin such as Cys-Ala-HSA
- a preferred C-terminal extension is the addition of Cys immediately adjacent the C-terminal of an albumin, such as a mature albumin.
- Cys is preferably immediately C-terminal to the last Leu (L585) residue.
- Such an albumin may be referred to as ‘albumin-Cys’, e.g. HSA-Cys.
- Other preferred C-terminal extensions of albumins such as SEQ ID No. 1 include albumin-Ala-Cys, such as HSA-Ala-Cys.
- Polypeptides suitable for providing extensions, as described above, may be added or inserted to the C- or, N-side of the C- or N-terminal amino acid of the albumin, such as to the C-side of L585 in SEQ ID No. 1.
- the polypeptide may or may not further comprise a further linker to which a conjugation partner, such as a bioactive compound, may be linked.
- a linker may comprise a primary amine such as a lysine.
- One or more conjugation competent cysteines may or may not be created in an albumin by insertion; for example by adding one or more additional cysteines without removal of an amino acid residue from the albumin sequence, or by substituting one or more adjacent amino acids with a larger number of residues containing at least one cysteine, thus extending the overall length of the polypeptide.
- a cysteine residue may be introduced immediately adjacent an albumin residue identified herein.
- the cysteine residue may be introduced as a single cysteine residue or within a polypeptide.
- the polypeptide may be from 2 to 50 amino acids long, preferably from 2, 10, 20, 30, or 40 to 10, 20, 30, 40 or 50 amino acids long.
- the invention includes substitution of one of the cysteine residues in one or more disulphides bond of an albumin with a different amino acid residue, so breaking the disulphide bond to leave an additional free thiol group.
- a cysteine of one or more of the 17 naturally occurring disulphide bonds of HSA may be substituted to provide a conjugation-competent cysteine. Such a substitution causes the cysteine which has not been substituted to no longer have a disulphide binding partner and therefore provide a free thiol group.
- Conjugation competent cysteines may be provided from one or more of the naturally occurring disulphide bonds of an albumin such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the naturally occurring disulphide bonds of an albumin such as HSA (e.g. SEQ ID No. 1).
- an albumin such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the naturally occurring disulphide bonds of an albumin such as HSA (e.g. SEQ ID No. 1).
- one cysteine residue which naturally forms a disulphide bond with another cysteine residue may or may not be substituted with a relatively conserved amino acid residue, particularly Ser, Thr, Val or Ala.
- cysteine residues involved in disulphide bonding are C53, C62, C75, C91, C90, C101, C124, C169, C168, C177, C200, C246, C245, C253, C265, C279, C278, C289, C316, 0361, C360, C369, C392, C438, C437, C448, C461, C477, C476, C487, C514, C559, C558 and C567. Cysteine residues preferred for modification (i.e.
- deletion or substitution may in particular correspond to C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567 thus generating a conjugation competent cysteine at one or more of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 of SEQ ID No. 1.
- conjugation competent cysteines may or may not be created in albumin by deletion of one of the cysteines of a disulphide bond in the protein structure, so breaking the disulphide bond to provide an additional free thiol group.
- one or more of the cysteine residues present in the albumin molecule, but not involved in disulphide bonding may or may not be deleted (i.e. without substitution with a different amino acid) or may or may not be substituted with a different amino acid, particularly Ser, Thr, Val or Ala.
- the conjugation competent cysteine residues when the polypeptide is folded, may or may not be relatively evenly distributed over the surface of the folded protein.
- folded includes folding of a polypeptide/protein into its natural configuration, for example the most thermodynamically stable folded configuration.
- An advantage of relatively even distribution is that it allows conjugation of two or more moieties to the thio-albumin without steric hindrance between two or more of the conjugated moieties. This has the advantage of minimising, and optionally eliminating, potential loss of activity due to issues such as steric hindrance between adjacent moieties (conjugation partners) which may be conjugated to the thio-albumin.
- moieties for example bioactive molecules, may be relatively bulky.
- the two or more conjugation competent cysteines are distributed over the surface of the thio-albumin molecule such that they are spaced as far from each other as possible, for example geometrically possible.
- the distance between two or more conjugation competent cysteines is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms.
- each conjugation competent cysteine is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms distant from all other conjugation competent cysteines in the molecule.
- the distance between two conjugation competent cysteines is preferably a distance which is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule, for example as shown in a model of an albumin.
- the longest axis of SEQ ID No. 1 as shown in protein structure 1AO6 is approximately 85 Angstroms. Therefore, it is preferred that the two or more of the cysteine residues are at least 65, 70, 75 or most preferably 80 Angstroms apart.
- Most preferably each conjugation competent cysteine residue is at a distance of at least 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule.
- the side chains of conjugation competent cysteines are directed away from each other and/or directed so that a moiety conjugated to the cysteine will be directed away from the centre of the albumin structure. This provides the advantage of preventing interactions between the conjugated moieties and/or the albumin moiety itself.
- candidate amino acid residues may be visually inspected using software such as PyMOL (Warren L. DeLano “The PyMOL Molecular Graphics System” DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org).
- Candidate amino acids may be divided into categories based on their proximity to other members of that group. For example, candidate amino acids may be divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 categories. It is preferred that combinations of candidate amino acids are selected from different categories. That is, it is preferred that a thio-albumin contains one or fewer mutations from each category.
- Selection Groups 1 and 2 correspond to the selection criteria (A) and (B) (respectively) from FIGS. 5A and 5B of the selection method described herein.
- Selection Group 3 corresponds to the residues identified in FIG. 6B .
- Particularly favoured residues are given in FIG. 6A and 6B in which the column headings are the same as those in FIG. 1 with the addition of ‘Selection Group’ and ‘Proximity Group’ as described herein.
- Proximity Group allocation of a proximity group as described herein to describe subsets of sites within HSA (specifically SEQ ID No. 1).
- candidate amino acid residues selected in Selection Group 1 were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of Selection Group 1.
- Five groups were generated (labeled A to E in FIG. 10 ‘proximity group’, right hand column), four were generated by visual inspection.
- Group E contains amino acid residues not visible in 1AO6 which are known to be in the N-terminal region.
- cys-34 is present in the proximity group A.
- amino acid residues selected in Selection Group 2 were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of Selection Group 2. Five groups were generated (labeled F-J in FIG. 10 ‘proximity group’, right hand column)
- the preferred free cysteine residues selected in Selection Group 3 (listed in FIG. 6B ), which can be generated by mutations causing disruption of disulphide bonds were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of that group. Four groups were generated (labeled K-N in FIG. 10 ‘proximity group’, right hand column). When referring to the residues of selection group 3, the cited residues are the resultant conjugation competent cysteines (e.g. FIG. 6B ). In order to generate such a conjugation competent cysteine it is clear that the counterpart cysteine (e.g. FIG. 6A ) in the disulphide bond should be removed for example by deletion or substitution.
- amino acid residues which occur in different ‘proximity’ groups may be preferred over those that occur within the same proximity group.
- there are 14 proximity groups i.e. A to N). It is preferred that, for a thio-albumin having two or more conjugation competent cysteines, there is zero or one conjugation competent cysteine defined from each of the 14 groups. That is, it is preferred that such a thio-albumin does not contain two or more conjugation competent cysteines falling within the same group. A large number of permutations exist which meet this criterion.
- T79+A364 in which one residue is selected from proximity group A to combine with A364 in proximity group C, would be preferred over T79+E86 which both occur in proximity group A.
- proximity groups A, F or K For combinations including cysteine-34, it is preferred not to select residues from proximity groups A, F or K. That is, it is preferred to select residues from one or more of proximity groups B to E, G to J and L to N.
- Examples of preferred mutations selected from within Selection Group 1 may include the following:
- Examples of preferred mutations selected from within Selection Group 2 may include the following:
- Examples of preferred site selected from within Selection Group 3 for the conjugation competent free-thiols may include the following:
- Combinations of sites from Selection Groups 1, 2 and 3 can also be made, where sites from Selection Group 1 are typically preferred to sites from Selection Group 2, which are typically preferred to sites selected from Selection Group 3.
- Examples of sites from Selection Groups 1+2 may include residues from proximity groups C+I, such as A364+A581.
- residues from proximity groups A+G+I, such as C34+S270+A581, from proximity groups A+H+G+I, such as C34+D129°S270+A581, from proximity groups A+C+I, such as T79+A364+A581, or residues from proximity groups C+I+H such as A364+A581+D129 are also preferred.
- Examples of sites from Selection Groups 1+3 may include residues from proximity groups A+L+M, such as C34+C169+C316, from proximity groups C+L, such as A364+C177 are preferred.
- residues from proximity groups B+M, such as D562+C369 are preferred.
- Examples of sites from Selection Groups 2+3 may include residues from proximity groups H+M, such as D129+C369 are preferred. Alternatively, residues from proximity groups I+M, such as A581+C369 are preferred.
- Examples of sites from Selection Groups 1+2+3 may include residues from proximity groups A+H+M+D, such as C34+D129+C360+L585, from proximity groups B+H+M, such as D562+D129+C369 are preferred.
- the above albumin variants of the invention may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position, if based on an albumin other than SEQ ID No. 1.
- a preferred thio-albumin comprises SEQ ID No. 1 with Cys at positions 2 and 585 in addition to the naturally occurring Cys at position 34 (SEQ ID No. 78, construct ‘TA33’).
- a more preferred thio-albumin comprises SEQ ID No. 1 with Cys at positions 2, 364, 562, 585 in addition to the naturally occurring Cys at position 34 (SEQ ID No. 82, construct ‘TA38’).
- Thio-albumins comprising three or four of the Cys at positions 2, 364, 562 and 585 may also be preferred.
- the polypeptide may or may not comprise at least one mutation that reduces glycosylation.
- a third aspect of the invention provides a polynucleotide which encodes the polypeptide according to the invention.
- the polynucleotide may or may not be codon-optimised relative to the host from which it is to be expressed.
- SEQ ID No. 2 provides the usual coding sequence of HSA (SEQ ID No. 1).
- SEQ ID No. 3 provides a coding sequence of HSA (SEQ ID No. 1) which is codon-optimised for expression from S. cerevisiae .
- SEQ ID No. 2 or 3 may be mutated in order to provide a polynucleotide which encodes a polypeptide according to the invention.
- the polynucleotide is synthetic and/or recombinant.
- the polynucleotide is an isolated polynucleotide.
- the polynucleotide may encode an HSA with or without a leader sequence.
- the polynucleotide may encode an HSA with the natural leader sequence of HSA (amino acids 1 to 24 of SEQ ID No. 102) or an HSA with a fusion leader sequence (amino acids 1 to 24 of SEQ ID No. 49).
- a fourth aspect of the invention provides a plasmid comprising the polynucleotide of the third aspect of the invention.
- the plasmid may be a 2 micron based plasmid such as those described in WO2005/061719, WO2005/061718 and WO2006/067511 (all incorporated herein by reference).
- the plasmid may exhibit enhanced chaperone activity, for example through over expression of a chaperone, particularly PDI.
- a fifth aspect of the invention provides an expression system such as a host cell comprising a polynucleotide according to the third aspect of the invention and/or a plasmid of the fourth aspect of the invention.
- the host cell is a mammalian cell such as a human or bovine cell, or a fungal cell such as a yeast cell.
- the host cell may be a bacterial cell such as a Bacillus or Escherichia coli or a viral cell such as Baculovirus or a plant cell such as a rice e.g. Oryza sativa .
- the cell is a yeast cell such as a Saccharomyces (e.g. S. cerevisiae ), a Pichia or an Aspergillus cell.
- a sixth aspect of the invention provides a conjugate which comprises a conjugation partner, such as a bioactive compound, and a polypeptide according to the invention, wherein the conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide.
- the conjugation partner may be a therapeutic, diagnostic or imaging compound such as those mentioned herein.
- the conjugate may comprise 2 or more, for example 2, 3, 4, 5, 6, 7,8, 9 or 10, conjugation partners which may each be different and/or may be multiple copies of the same compound.
- each conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide, however conjugation partners may be linked by other means for example by a genetic fusion or covalent bonds to non-cysteine amino acids such as lysine.
- a seventh aspect of the invention provides a method of producing a polypeptide of the invention comprising:
- the present invention also provides a method for producing a polypeptide (or protein) of the invention, the method comprising: (a) providing a host cell of the invention comprising a polynucleotide encoding protein product of choice as defined above; and (b) growing the host cell (for example, culturing the host cell in a culture medium); thereby to produce a cell culture or recombinant organism comprising an increased level of the protein product of choice compared to the level of production of the protein product of choice achieved by growing (for example, culturing), under the same conditions, the same host cell that has not been genetically modified to cause over-expression of one or more helper proteins.
- the step of growing the host cell may or may not involve allowing a host cell derived from a multicellular organism to be regrown into a multicellular recombinant organism (such as a plant or animal) and, optionally, producing one or more generations of progeny therefrom.
- the method may or may not further comprise the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.
- the production method may comprise linking a conjugation partner to the polypeptide of the invention through a conjugation competent cysteine residue of the polypeptide. Suitable conjugation methods and conjugation partners are described herein.
- An eighth aspect of the invention provides a composition comprising a conjugate according to the invention and at least one pharmaceutically acceptable carrier and/or diluent.
- a ninth aspect of the invention provides a method for making a pharmaceutical ingredient and/or a pharmaceutical product comprising making a thio-albumin according to the present invention, optionally conjugating a further molecule to the thio-albumin, optionally formulating the resultant conjugate with a pharmaceutically acceptable diluent and/or carrier and optionally preparing the product in unit dosage form.
- a tenth aspect of the invention provides use of a polypeptide according to the invention for the production of a thio-albumin-conjugate.
- An eleventh aspect of the invention provides use of a conjugate according to the invention and/or produced by a method according to the invention for treatment of disease, treatment of illness and/or diagnosis.
- a twelfth aspect of the invention provides a gel comprising one or more albumins according to the invention.
- the gel may be formed by any suitable method.
- the gel may be formed by incubating an albumin solution, or suspension, at a suitable temperature e.g. room temperature (15 to 25° C., such as 20° C.) or body temperature (36 to 38° C., preferably 36.9° C.).
- a gel may be used to coat medical devices, such as a stent.
- a gel may be used in or on a wound dressing.
- the albumin may be applied to the medical device or wound dressing before or after it has gelled.
- the albumin may be applied ex situ or in situ (e.g. applied to a medical device or dressing before, after or during its application on to or insertion into a human or animal body).
- the polypeptides and/or conjugates of the invention may be used to prepare nanoparticles which may be used, for example, in angiogenic applications, anti-angiogenic applications and to coat a medical device such as a stent.
- Nanoparticles are effective at targeting, for example to non tight-junctions, and therefore can be useful for targeting tumours such as cancerous tumours.
- Nanoparticles can also be useful to target antigen in order to provoke an immune response since nanoparticles are particularly susceptible to engulfment and presentation by phagocytes.
- the invention provides nanoparticles consisting only of thio-albumin according to the invention which may or may not be conjugated to a moiety (conjugation partner).
- the invention also provides nanoparticles comprising thio-albumin according to the invention, which may or may not be conjugated to a moiety, and one or more other constituents of a nanoparticle which may or may not be albumin related.
- a thio-albumin according to the invention comprises at least two conjugation competent cysteine residues located on the surface of the polypeptide.
- Such a thio-albumin may be used for the preparation of nanoparticles in which one or more conjugation competent cysteine residues may be used in the formation of a nanoparticle and one or more conjugation competent residue is used for conjugation to a conjugation partner, for example to a bioactive molecule.
- the invention relates to all albumins. Whilst preferred residues have been identified in relation to SEQ ID No. 1, the skilled person would be able to identify equivalent residues in other albumin sequences, such as the albumins disclosed in FIGS. 2 and 3 , and understand that mutations of albumins (other than SEQ ID No. 1) at such equivalent residues are part of the invention. Equivalent residues can be identified by, for example, homology alignment with SEQ ID No. 1. A residue in an albumin other than SEQ ID No. 1 may or may not have an identical residue coordinate to its equivalent residue in SEQ ID No. 1. Thus the invention provides thio-albumins based on any albumin sequence, such as the sequences shown in Table 1 and, more preferably, those shown in FIG. 2 and/or 3 . ‘Based on’ includes modification of an albumin sequence to introduce one or more additional free-thiols.
- Recombinant albumins can offer advantages over animal-derived albumins by having a higher level of conjugation-competent free thiol groups,' and can be manufactured without the risk of contamination with pathogenic prions and viruses.
- An advantage of a thio-albumin conjugate is that the thio-albumin part may be prepared separately to a conjugation partner. Therefore, one batch of thio-albumin may be used to produce many different thio-albumin conjugates.
- the individual components of the conjugate can be manufactured by different methods and therefore are not restricted to a single method, such as heterologous protein expression in a host cell such as a yeast.
- a thio-albumin may comprise multiple conjugation sites and therefore a single thio-albumin may be conjugated to more than one type of conjugation partner (e.g. therapeutic agent, diagnostic agent, targeting agent, imaging agent) and/or to multiple copies of one or more types of conjugation partner.
- conjugation partner e.g. therapeutic agent, diagnostic agent, targeting agent, imaging agent
- the ability to conjugate the thio-albumin to different types of conjugation partners allows the provision of a multi-functional species.
- the ability to conjugate the thio-albumin to multiple copies of a conjugation partner allows the concentration of molecule to be increased and therefore increase the amount, or volume, of thio-albumin conjugate required for a given purpose relative to a conjugate having only a single copy of the conjugation partner.
- Advantages of delivering drugs via an albumin fusion protein are discussed in Osborn, et al. (2002). J Pharmacol Exp Ther 303(2): 540-8. It is expected that delivery of drugs via a conjug
- FIG. 1 gives the amino acid residues for 1AO6.
- albumins relate to all albumins and their structures. Structures of albumin are available to the skilled person, for example the atomic coordinates for the tertiary structure of human albumin are available at the GenBank DNA database at www.ncbi.nlm.nih.gov.
- the albumin used in the invention may be a naturally occurring albumin, an albumin-related protein or a variant thereof such as a natural or engineered variant. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins.
- An albumin, of this invention may comprise the sequence of an albumin protein obtained from any source. Typically the source is mammalian such as human or bovine.
- the serum albumin is human serum albumin (“HSA”).
- HSA human serum albumin
- the term “human serum albumin” includes a serum albumin having an amino acid sequence naturally occurring in humans, and variants thereof.
- the albumin has the amino acid sequence of SEQ ID No. 1 or a variant or fragment thereof, preferably a functional variant or fragment thereof.
- the HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 0 073 646 and EP 0 286 424.
- a fragment or variant may or may not be functional.
- a fragment or variant may retain the ability to bind to an albumin receptor such as FcRn to at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the ability of the parent albumin (from which the fragment or variant derives) to bind to the receptor.
- Relative binding ability may be determined by methods known in the art such as surface plasmon resonance studies.
- the albumin may be a naturally-occurring polymorphic variant of human albumin or of a human albumin analogue. Generally, variants or fragments of human albumin will have at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, (preferably at least 80%, 90%, 95%, 100%, 105% or more) of human albumin's ligand binding activity (for example FcRN-binding), mole for mole.
- human albumin's ligand binding activity for example FcRN-binding
- the “albumin” may comprise the sequence of bovine serum albumin.
- bovine serum albumin includes a serum albumin having an amino acid sequence naturally occurring in cows, for example as taken from Swissprot accession number P02769, and variants thereof as defined herein.
- bovine serum álbumin also includes fragments of full-length bovine serum albumin or variants thereof, as defined herein.
- the albumin may comprise the sequence of an albumin derived from one of serum albumin from dog (e.g. see Swissprot accession number P49822-1), pig (e.g. see Swissprot accession number P08835-1), goat (e.g. as available from Sigma as product no. A2514 or A4164),), cat (e.g. see Swissprot accession number P49064-1), chicken (e.g. see Swissprot accession number P19121-1), ovalbumin (e.g. chicken ovalbumin) (e.g. see Swissprot accession number P01012-1), turkey ovalbumin (e.g. see Swissprot accession number O73860-1), donkey (e.g.
- Swissprot accession number Q5XLE4-1 guinea pig(e.g. see Swissprot accession number Q6WDN9-1), hamster (see DeMarco et al. (2007). International Journal for Parasitology 37(11): 1201-1208), horse (e.g. see Swissprot accession number P35747-1), rhesus monkey (e.g. see Swissprot accession number Q28522-1), mouse (e.g. see Swissprot accession number P07724-1), pigeon (e.g. as defined by Khan et al, 2002, Int. J. Biol. Macromol., 30(3-4),171-8), rabbit (e.g.
- Swissprot accession number P49065-1 see Swissprot accession number P49065-1
- rat e.g. see Swissprot accession number P02770-1
- sheep e.g. see Swissprot accession number P14639-1 and includes variants and fragments thereof as defined herein.
- the albumin may comprise the sequence of an albumin such as a serum albumin or an ovalbumin, for example those shown in Table 1, below, and includes variants and fragments thereof as defined herein.
- albumin Many naturally occurring mutant forms of albumin are known. Many are described in Peters, (1996, All About Albumin: Biochemistry, Genetics and Medical Applications , Academic Press, Inc., San Diego, Calif., p.170-181). A variant as defined herein may be one of these naturally occurring mutants such as those described in Minchiotti at al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768,.
- a “variant albumin” refers to an albumin protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property, for example binding activity (type of and specific activity e.g.
- a fatty acid such as a long-chain fatty acids, for exampleoleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and/or palmitoleic (C16:1)), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed.
- “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein, e.g. the protein from which the variant is derived. Such characteristics may be used as additional selection criteria in the invention.
- an albumin variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with a naturally occurring albumin such as SEQ ID No. 1.
- the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
- the alignment may alternatively be carried out using the Clustal W program or the Clustal V program and therefore allow calculation of % homology between sequences of a multiple alignment and/or calculation of % identity between sequences of a pairwise alignment.
- the parameters used may be as follows:
- Clustal V Pairwise alignment parameters: Ktuple: 1, Gap Penalty: 3, Window: 5, Diaganols: 5; Multiple alignment parameters: Gap penalty 10, gap length penalty 10.
- conservative amino acid substitutions refers to substitutions made within the same group, and which typically do not substantially affect protein function.
- conservative substitutions is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
- Such variants may be made by techniques well known in the art, such as by site-directed mutagenesis as disclosed in U.S. 4 Pat. No 4,302,386 issued 24 Nov. 1981 to Stevens, incorporated herein by reference.
- the Venn diagram of FIG. 4 may be used to determine conservative amino acid substitutions: Using FIG. 4 ., a conservation mutation score (ranging from 0 to 5) may be calculated. A score of 0 is the highest conservation, which, for cysteine, is only assigned for substitution of a cysteine residue with another cysteine residue. For changes from any other amino acid to a cysteine, the score may be 1, 2, 3, 4, 5. A score of 1 is a more conservative substitution that a score of 2, 3, 4 or 5. A score of 5 is assigned to the lowest conservation between a substituted amino acid and the cysteine. The score of 0 to 5 is calculated from FIG. 4 as the number of boundaries (i.e. lines) crossed to go from cysteine to the appropriate amino acid. Thus the score for cysteine is 0 as no boundaries are crossed. Likewise, the score of aspartic acid (D) is 3, since 3 boundaries are crossed.
- D aspartic acid
- these scores are provided for each of the amino acid residues in the column labelled ‘Conserved Mutation to Cysteine’.
- aspartic acid methionine, proline, glutamine, valine, tryptophan, tyrosine, glycine, asparagine, alanine, serine and threonine are preferred since they are relatively conserved with cysteine. More preferred are those amino acids with a score of 2 or less i.e. glycine, asparagine, alanine, serine, threonine. Most preferred are those with a score of 1, i.e. alanine, serine, threonine.
- residues with a score of 4 or more i.e. glutamic acid, phenylalanine, isoleucine, lysine, leucine, histidine and arginine are less preferred and may not be preferred at all.
- “conservative” amino acid substitutions refers to substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
- basic amino acids such as arginine, lysine, histidine
- acidic amino acids such as glutamic acid and aspartic acid
- polar amino acids such as glutamine and asparagine
- hydrophobic amino acids such as leucine, isoleucine, valine
- aromatic amino acids such as phenylalanine, tryptophan, tyrosine
- a conservative substitution of alanine-2 in SEQ ID No 1 can include glycine or serine.
- Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group.
- a non-conservative substitution could include the substitution of a polar amino acid for a hydrophobic amino acid.
- fragment includes any fragment of full-length albumin or a variant thereof, so long as at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein. A fragment will typically be at least 50 amino acids long. A fragment may comprise at least one whole sub-domain of albumin.
- HSA HSA proteins
- domain I was defined as consisting of amino acids 1-197
- domain II was defined as consisting of amino acids 189-385
- domain III was defined as consisting of amino acids 381-585. Partial overlap of the domains occurs because of the extended a-helix structure (h10-h1) which exists between domains I and II, and between domains II and III (Peters, 1996, op. cit., Table 2-4).
- HSA also comprises six sub-domains (sub-domains IA, IB, IIA, IIB, IIIA and IIIB).
- Sub-domain IA comprises amino acids 6-105
- sub-domain IB comprises amino acids 120-177
- sub-domain IIA comprises amino acids 200-291
- sub-domain IIB comprises amino acids 316-369
- sub-domain IIIA comprises amino acids 392-491
- sub-domain IIIB comprises amino acids 512-583.
- a fragment may comprise a whole or part of one or more domains or sub-domains as defined above, or any combination of those domains and/or sub-domains.
- a fragment may comprise or consist of at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of an albumin or of a domain of an albumin.
- single or multiple heterologous fusions comprising any of the above; or single or multiple heterologous fusions to albumin, or a variant or fragment of any of these may be used.
- Such fusions include albumin N-terminal fusions, albumin C-terminal fusions and co-N-terminal and C-terminal albumin fusions as exemplified by WO 01/79271.
- FIGS. 2 and 3 show alignments of various albumin family proteins with HSA (SEQ ID NO: 1), identified as ‘P02768’.
- the protein sequences include the albumin leader sequence. These alignments can be used to identify conserved regions and amino acid residues corresponding to those in HSA selected as described above. One or both alignments can also be used to assign a homology score to an amino acid residue in an albumin sequence.
- the Clustal V method may be used (above).
- the alignment of two amino acid sequences may also be determined by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0.
- the Needle program implements the global alignment algorithm described in Needleman and Wunsch (1970) “A general method applicable to the search for similarities in the amino acid sequence of two proteins.” J. Mol. Biol. 48, 443-453.
- the substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.
- FIG. 2 shows an alignment of sixteen mammalian albumin family proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal W.
- the protein sequences include the albumin leader sequence. Each sequence is labelled with the animal from which it derives and its database accession number.
- FIG. 3 shows alignments of thirty three albumin family (both mammalian and non-mammalian) proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal V.
- the protein sequences include the albumin leader sequence.
- Homology may be determined with reference to FIG. 2 and/or FIG. 3 .
- the degree of identity between a given amino acid sequence and SEQ ID NO: 1 may be calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the shorter of the two sequences. The result may be expressed in percent identity.
- An exact match occurs when the two sequences have identical amino acid residues in the same positions of the overlap.
- the length of a sequence is the number of amino acid residues in the sequence.
- FIG. 1 column labelled ‘Align 1 (Mamm. W ('mammalian, Clustal W)) provides the homology level for each position of SEQ ID No. 1 as calculated by the alignment of mammalian albumins given in FIG. 2 .
- the homology level score may be calculated.
- a score of 100 is the highest conservation and indicates there are no changes at that residue when the sequence from human serum albumin is compared with other mammalian albumin sequence, whereas a score of 0 indicates the lowest level of conservation between the aligned sequences.
- the homology level score for each amino acid residue in HSA ( FIG. 1 , column labelled ‘Align 2 (Var. Sps. (‘various species (i.e. mammalian and non-mammalian), Clustal V’)) calculated using the strength of the histogram provided by Megalign when various albumins (including non-mammalian albumins) are aligned using Clustal V.
- Align 2 Var. Sps. (‘various species (i.e. mammalian and non-mammalian), Clustal V’)
- a person skilled in the art will appreciate that a range of different albumin sequences and alignment algorithms may be used to calculate the homology level score.
- preferred residues include those which are not highly conserved (for example those with a score of less than 40, more preferably less than 20 and most preferably 0) are preferred and those with a higher level of homology (for example those with a score of more than 40, more than 60, more than 80 and most preferably 100) are less preferred.
- Each of the amino acid residue in HSA (SEQ ID No. 1) were scored according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with mammalian albumins ( FIG. 1 , column labelled ‘Adj. 100%'s (Align 1). This is because if an amino acid is within a highly conserved domain, it may be important to the structure of function of the protein and thus disruption may be undesirable.
- a score of 0 indicates the residue is not adjacent to any residue with 100% homology (with reference to the alignment of FIG.
- Residues with a score of 0 or 1 are preferred. Residues with a score of 0 are most preferred.
- amino acid residues with a score of 2 are preferably deselected using the method of the invention since these amino acid residues were assumed to occur in a region of high homology which would be unlikely to accept a mutation to an alternative amino acid.
- phenylalanine-11 is adjacent to one 100% conserved residue, in a region of conserved residues, and is less preferred to a residue, such as alanine-2 (A2), which has no adjacent 100% conserved residues.
- mouse albumin contains 36 cysteine residues, all the cysteines involved in disulphide bonding (by homology to HSA) are present, as is cysteine-34, however a cysteine residue is present at 579 on mature mouse protein but not other mammalian albumin sequences therefore thio-albumin mutein S579C may be preferred as its lack of homology with other mammalian albumins suggests that it may not be particularly important to the structure and/or function of this albumin.
- FIG. 2 shows that compared to other mammalian albumins, gerbil albumin has an additional alanine residue between alanine-2 (A2) and histidine-3 (H3), indicating that insertion of a cysteine residues after residue 2 (e.g. A2 of SEQ ID No. 1) and before residue 3 (e.g. H3 of SEQ ID No. 1) is preferred.
- A2 alanine-2
- H3 histidine-3
- guinea pig albumin has a serine residue at cysteine-34 (C34).
- C34S cysteine-34
- Most mammalian albumin sequences (with the exception of human serum albumin) have a sequence which is less than or equal to 584 amino acids in length (less than or equal to 608 amino acids including leader sequence).
- the additional amino acid residue present on human serum albumin appears to be at the C-terminus without any cognate alignment amino acid residues in the other mammalian serum albumin sequences.
- a thio-albumin variant containing G584C and a deletion of L585 may be preferred.
- albumin sequences (Bovine, Donkey, Goat, Horse, Sheep, Pig)are 583 amino acids in length (607 amino acids including leader sequence). Using the alignment in FIG. 2 , it can be seen that these species albumin sequences do not have a residue corresponding R117 (R141 including leader sequence) therefore a thio-albumin containing V116C and a deletion of R117 or a thio-albumin containing a deletion of R117 and P118C may be preferred. In such a thio-albumin the length of the amino acid sequence would be reduced relative to SEQ ID No. 1.
- the albumin variant may have at least 40% identity with SEQ ID NO: 1, particularly at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity.
- the thio-albumin may optionally be fused to one or more conjugation partners for example through a genetic or chemical fusion.
- the fusion may be at the N- or C-terminus or comprise an insertion.
- the open reading frame of any other gene or variant, or part or either can be utilised as an open reading frame for use with the present invention.
- the open reading frame may encode a protein comprising any sequence, be it a natural protein (including a zymogen), or a variant, or a fragment (which may, for example, be a domain) of a natural protein; or a totally synthetic protein; or a single or multiple fusion of different proteins (natural or synthetic).
- Such proteins can be taken, but not exclusively, from the lists provided in WO 01/79258, WO 01/79271, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, or a variant or fragment thereof; the disclosures of which are incorporated herein by reference.
- these patent applications present the list of proteins in the context of fusion partners for albumin, the present invention is not so limited and, for the purposes of the present invention, any of the proteins listed therein may be presented alone or as fusion partners for albumin or any other protein or fragment or variant of any of the above, as a desired polypeptide.
- Examples of chemical fusions (also known as conjugations) of albumin are given in Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; and Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8.
- An advantage of using a genetically or chemically fused albumin is that either or all of the molecules which contribute to the fusion may have improved properties relative to the unfused molecule(s) (Balan et al. (2006) Antivir Ther 11(1): 35-45).
- Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz, F. (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused conjugation partner, for example bioactive molecule, from inactivation.
- the polypeptide may display modified (e.g. reduced) glycosylation, such as, but not limited to reduced N-linked glycosylation or reduced O-linked glycosylation.
- the N-linked glycosylation pattern of an albumin molecule can be modified by adding/removing amino acid glycosylation consensus sequences such as N-X-S/T, at any or all of the N, X, or S/T position.
- Albumin polymorphisms exist with N-linked glycosylation.
- Albumin mutants may have recycling time such that the efficacy of a mutant as a bioactive carrier is improved.
- Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell.
- rHA recombinant human albumin
- yeast species can be modified by O-linked glycosylation, generally involving mannose.
- the mannosylated albumin is able to bind to the lectin Concanavalin A.
- the amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced.
- the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
- disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia pastoris or Kluyveromyces lactis .
- the sequence of PMT1 (or any other PMT gene) isolated from S. cerevisiae may be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species.
- the cloning of the PMT1 homologue of Kluyveromyces lactis is described in WO 94/04687.
- the step of “purifying the thus expressed heterologous protein from the cultured host cell or the culture medium” optionally comprises cell immobilization, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilization, separation and/or breakage.
- Cell immobilization techniques such as encasing the cells using calcium alginate beads, are well known in the art.
- cell separation techniques such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like are well known in the art.
- methods of cell breakage including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
- the at least one other purification step may be any other step suitable for protein purification known in the art.
- purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference.
- thio-albumin or fusions of thio-albumin and another protein or proteins can be prepared by methods know to the art (Sanker, (2004), Genetic Eng. News, 24, 22-28, Schmidt, (2004), Appl. Microbiol. Biotechnol., 65, 363-372) including but not limited to expression in mammalian cell culture (Mason et al., (2004), Protein Expr.
- the host cell may be any type of cell.
- the host cell may or may not be an animal (such as mammalian, avian, insect, etc.), plant (such as Oryza sativa ), fungal or bacterial cell.
- Bacterial and fungal, such as yeast, host cells may or may not be preferred.
- Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).
- a typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
- An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, N.J., USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
- Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls.
- One such method involves ligation via cohesive ends.
- Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
- a further method uses synthetic double stranded oligonucleotide linkers and adaptors.
- DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3′ termini and fill in recessed 3′ ends.
- Synthetic linkers and pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini.
- Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end.
- a DNA fragment or DNA fragments can be ligated together by the action of DNA ligase in the presence or absence of one or more synthetic double stranded oligonucleotides optionally containing cohesive ends.
- Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Sigma-Genosys Ltd, London Road, Pampisford, Cambridge, United Kingdom.
- thio-albumin or fusions of thio-albumin and another protein or proteins may be expressed from a nucleotide sequence, which may or may not contain one or more introns. Additionally the nucleotide sequence may or may not be codon optimised for the host by methods known to the art.
- the thio-albumin or fusions of thio-albumin and another protein or proteins can be expressed as variants with reduced N-linked glycosylation.
- HSA human serum albumin
- Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell.
- the mannosylated albumin would be able to bind to the lectin Concanavalin A.
- the amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687).
- the most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may or may not be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced.
- the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
- the yeast could be cultured in the presence of a compound that inhibits the activity of one of the PMT genes (Duffy et al, “ Inhibition of protein mannosyltransferase 1 ( PMT 1) activity in the pathogenic yeast Candida albicans ”, International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, 26-31 August 2001, Monte Verita, Switzerland, Poster Abstract P38; the poster abstract may be viewed at http://www.micro.biol.ethz.ch/cellwall/). If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g.
- the yeast may or may not also have a deletion of the HSP150 and/or YAP3 genes as taught respectively in WO 95/33833 and WO 95/23857.
- the HSA variant may be produced by recombinant expression and secretion.
- yeast such as Saccharomyces cerevisiae
- suitable promoters for S. cerevisiae include those associated with the PGK1 gene, GAL1 or GAL10 genes, TEF1, TEF2, PYK1, PMA1, CYC1, PHO5, TRP1, ADH1, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, a-mating factor pheromone, a-mating factor pheromone, the PRB1 promoter, the PRA1 promoter, the GPD1 promoter, and hybrid promoters involving hybrids of parts of 5′ regulatory regions with parts of
- Suitable transcription termination signals are well known in the art. Where the host cell is eukaryotic, the transcription termination signal is preferably derived from the 3′ flanking sequence of a eukaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3′ flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different. In that case, and where the host is a yeast, preferably S. cerevisiae , then the termination signal of the S. cerevisiae ADH1, ADH2, CYC1, or PGK1 genes are preferred.
- the promoter and open reading frame of the gene encoding the recombinant protein comprising the sequence of an albumin mutant may be flanked by transcription termination sequences so that the transcription termination sequences are located both upstream and downstream of the promoter and open reading frame, in order to prevent transcriptional read-through into any neighbouring genes, such as 2 ⁇ m genes, and vice versa.
- the favoured regulatory sequences in yeast include: a yeast promoter (e.g. the Saccharomyces cerevisiae PRB1 promoter), as taught in EP 431 880; and a transcription terminator, preferably the terminator from Saccharomyces ADH1, as taught in EP 60 057.
- a yeast promoter e.g. the Saccharomyces cerevisiae PRB1 promoter
- a transcription terminator preferably the terminator from Saccharomyces ADH1, as taught in EP 60 057.
- non-coding region may incorporate more than one DNA sequence encoding a translational stop codon, such as UAA, UAG or UGA, in order to minimise translational read-through and thus avoid the production of elongated, non-natural fusion proteins.
- a translational stop codon such as UAA, UAG or UGA
- the recombinant protein comprising the sequence of an albumin mutant is secreted.
- a sequence encoding a secretion leader sequence may be included in the open reading frame.
- a polynucleotide according to the present invention may comprise a sequence that encodes a recombinant protein comprising the sequence of an albumin mutant operably linked to a polynucleotide sequence that encodes a secretion leader sequence.
- Leader sequences are usually, although not necessarily, located at the N-terminus of the primary translation product of an ORF and are generally, although not necessarily, cleaved off the protein during the secretion process, to yield the “mature” protein.
- the term “operably linked” in the context of leader sequences includes the meaning that the sequence that encodes a recombinant protein comprising the sequence of an albumin mutant is linked, at its 5′ end, and in-frame, to the 3′ end of a polynucleotide sequence that encodes a secretion leader sequence.
- the polynucleotide sequence that encodes a secretion leader sequence may be located, in-frame, within the coding sequence of the recombinant protein comprising the sequence of an albumin mutant, or at the 3′ end of the coding sequence of the recombinant protein comprising the sequence of an albumin mutant.
- leader sequences also called secretion pre regions and pre/pro regions
- Leader sequences direct a nascent protein towards the machinery of the cell that exports proteins from the cell into the surrounding medium or, in some cases, into the periplasmic space.
- a secretion leader sequence may be used for production of proteins in eukaryotic species such as the yeasts Saccharomyces cerevisiae, Zygosaccharomyces species, Kluyveromyces lactis and Pichia pastoris .
- This may comprise a signal (pre) sequence or a prepro leader sequence.
- Signal sequences are known to be heterogeneous in their amino acid sequence (Nothwehr and Gordon 1990, Bioessays 12, 479-484, or Gierasch 1989, Biochemistry 28, p923-930). In essence, signal sequences are generally N-terminally located, have a basic n-region, a hydrophobic h-region and a polar c-region.
- signal sequence will work, irrespective of the amino acid composition. How well they work, i.e. how much mature protein is secreted, depends upon the amino acid sequence.
- signal peptide is understood to mean a presequence which is predominantly hydrophobic in nature and present as an N-terminal sequence of the precursor form of an extracellular protein expressed in yeast.
- the function of the signal peptide is to allow the expressed protein to be secreted to enter the endoplasmic reticulum.
- the signal peptide is normally cleaved off in the course of this process.
- the signal peptide may be heterologous or homologous to the yeast organism producing the protein.
- Known leader sequences include those from the S.
- MF ⁇ -1 S. cerevisiae acid phosphatase protein
- HSA heat-shock protein-150
- MF ⁇ -1 S. cerevisiae mating factor alpha-1 protein
- HSA human serum albumin
- WO 90/01063 discloses a fusion of the MF ⁇ -1 and HSA leader sequences (also known as the fusion leader sequence (FL)).
- the natural albumin leader sequence may or may not be used to direct secretion of the recombinant protein comprising the sequence of an albumin mutant.
- any suitable plasmid may be used, such as a centromeric plasmid.
- the examples provide suitable plasmids (centromeric YCplac33-based vectors) for use to transform yeast host cells of the present invention.
- any other suitable plasmid may be used, such as a yeast-compatible 2 p m-based plasmid.
- Plasmids obtained from one yeast type can be maintained in other yeast types (Irie et al, 1991, Gene, 108(1), 139-144; Irie et al, 1991, Mol. Gen. Genet., 225(2), 257-265).
- pSR1 from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae .
- the plasmid may or may not be a 2 ⁇ m-family plasmid and the host cell will be compatible with the 2 ⁇ m-family plasmid used (see below for a full description of the following plasmids).
- a suitable yeast cell is Zygosaccharomyces rouxii ; where the plasmid is based on pSB1 or pSB2 then a suitable yeast cell is Zygosaccharomyces bailli ; where the plasmid is based on pSM1 then a suitable yeast cell is Zygosaccharomyces fermentati ; where the plasmid is based on pKD1 then a suitable yeast cell is Kluyveromyces drosophilarum ; where the plasmid is based on pPM1 then a suitable yeast cell is Pichia membranaefaciens ; where the plasmid is based on the 2 ⁇ m plasmid then a suitable yeast cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis .
- the plasmid may be based on the 2 ⁇ m plasmid and the yeast cell may be Saccharomyces cerevisiae .
- a 2 ⁇ m-family plasmid can be said to be “based on” a naturally occurring plasmid if it comprises one, two or preferably three of the genes FLP, REP1 and REP2 having sequences derived from that naturally occurring plasmid.
- Useful yeast episomal plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA), YEp24 (Botstein, D., et al. (1979) Gene 8, 17-24), and YEplac122, YEplac195 and YEplac181 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534).
- Other yeast plasmids are described in WO 90/01063 and EP 424 117, as well as the “disintegration vectors of EP-A-286 424 and WO2005061719.
- Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3, as are Ylplac204, Ylplac211 and Ylplac128 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534).
- Plasmids pRS413-416 are Yeast Centromere plasmids (YCps) as are YCplac22, YCplac33 and YCplac111 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534).
- helper also known as ‘chaperone’
- the host cell type may be selected for compatibility with the plasmid type being used.
- plasmids are disclosed in WO2005061719.
- Preferred helper proteins include PDI1, AHAI, ATP11, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1, CPR3, CPRE, DER1, DER3, DOA4, ERO1, EUG1, ERV2, EPS1, FKB2, FMO1, HCH1, HRD3, HSP10, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, KAR2, JEM1, MDJ1, MDJ2, MPD1, MPD2, PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SCJ1, SSA1, SSA2, SSA3, SSA4, SSBI, SSB2, SSC1, SSE2, SIL1, SLS1, ORM1, ORM2, PERI, PTC2, PSE1, UBC7, UBI4 and HAC
- Plasmids as defined herein may be introduced into a host through standard techniques. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual , Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. Methods for the transformation of S.
- Electroporation is also useful for transforming cells and is well known in the art for transforming fungal (including yeast) cell, plant cells, bacterial cells and animal (including vertebrate) cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
- a plasmid will transform not all of the hosts and it will therefore be necessary to select for transformed host cells.
- a plasmid may comprise a selectable marker, including but not limited to bacterial selectable marker and/or a yeast selectable marker.
- a typical bacterial selectable marker is the ⁇ -lactamase gene although many others are known in the art.
- Typical yeast selectable marker include LEU2, TRP1, HIS3, HIS4, URA3, URA5, SFA 1, ADE2, MET15, LYS5, LYS2, ILV2, FBA1, PSE1, PDI1 and PGK1.
- any gene whose chromosomal deletion or inactivation results in an unviable host can be used as a selective marker if a functional gene is provided on the plasmid, as demonstrated for PGK1 in a pgk1 yeast strain (Piper and Curran, 1990, Curr. Genet. 17, 119).
- Suitable essential genes can be found within the Stanford Genome Database (SGD), (http:://db.yeastgenome.org). Any essential gene product (e.g.
- auxotrophic (biosynthetic) requirement we include a deficiency which can be complemented by additions or modifications to the growth medium.
- essential marker genes in the context of the present application are those that, when deleted or inactivated in a host cell, result in a deficiency which cannot be complemented by additions or modifications to the growth medium.
- a plasmid may comprise more than one selectable marker.
- Transformed host cells may be cultured for a sufficient time and under appropriate conditions known to those skilled in the art, and in view of the teachings disclosed herein, to permit the expression of the helper protein(s) and the protein product of choice.
- the culture medium may be non-selective or place a selective pressure on the maintenance of a plasmid.
- prokaryotic host cells such as E. coli
- eukaryotic host cells such as mammalian cells
- Methods for culturing yeast are generally taught in EP 330 451 and EP 361 991.
- the thus produced protein product of choice may be present intracellularly or, if secreted, in the culture medium and/or periplasmic space of the host cell.
- the step of “purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium” optionally comprises cell immobilisation, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilisation, separation and/or breakage.
- Thio-albumin of the invention may be purified from the culture medium by any technique that has been found to be useful for purifying such proteins.
- cell separation techniques such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like) are well known in the art.
- methods of cell breakage including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
- the “at least one other purification step” may be any other step suitable for protein purification known in the art.
- purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference.
- Suitable methods include ammonium sulphate or ethanol precipitation, acid or solvent extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography (“HPLC”), reverse phase HPLC, conductivity adjustment and the like.
- HPLC high performance liquid chromatography
- the polypeptide may be purified to a commercially or industrially acceptable level of purity.
- commercially or industrially acceptable level of purity we include the provision of the thio-albumin and/or thio-albumin-conjugate in which other material (for example, one or more contaminants) are present at a level of less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% and, most preferably at a level of 0%.
- a commercially or industrially acceptable level of purity may be obtained by a relatively crude purification method by which the protein product of choice is put into a form suitable for its intended purpose.
- a protein preparation that has been purified to a commercially or industrially acceptable level of purity may, in addition to the protein product of choice, also comprise, for example, cell culture components such as host cells or debris derived therefrom.
- cell culture components such as host cells or debris derived therefrom.
- high molecular weight components such as host cells or debris derived therefrom
- the protein may or may not be purified to achieve a pharmaceutically acceptable level of purity.
- a protein has a pharmaceutically acceptable level of purity if it is essentially pyrogen free and can be used for its intended purpose and hence be administered in a pharmaceutically efficacious amount without causing medical effects not associated with the activity of the protein.
- the thio-albumin and/or thio-albumin-conjugate may be provided at a concentration of at least 10 ⁇ 4 g.L ⁇ 1 , 10 ⁇ 3 g.L ⁇ 1 , 0.01 g.L ⁇ 1 , 0.02 g.L ⁇ 1 , 0.03 g.L ⁇ 1 , 0.04 g.L ⁇ 1 , 0.05 g.L ⁇ 1 , 0.06 g.L ⁇ 1 , 0.07 g.L ⁇ 1 , 0.08 g.L ⁇ 1 , 0.09 g.L ⁇ 1 , 0.1 g.L ⁇ 1 , 0.2 g.L ⁇ l , 0.3 g.L ⁇ 1 , 0.4 g.L ⁇ 1 , 0.5 g.L ⁇ 1 , 0.6 g/L ⁇ 1 , 0.7 g.L ⁇ 1 , 0.8 g.L ⁇ 1 , 0.9 0 g.L ⁇ 1
- a method of the present invention may or may not further comprise the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
- a therapeutically useful protein obtained by a process of the invention is administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers or diluents.
- the carrier(s) or diluent(s) must be “acceptable” in the sense of being compatible with the desired protein.
- the carriers or diluents will be water or saline which will be sterile and pyrogen free.
- a method of the present invention may or may not further comprise the step of lyophilising the thus purified protein product of choice.
- the thio-albumin may be formulated by strategies given in “Protein Formulation and Delivery”, E. J. McNally (Ed.), published by Marcel Dekker Inc. New York 2000 and “Rational Design of Stable Protein Formulations-Theory and Practice”; J. F. Carpenter and M. C. Manning (Ed.) Pharmaceutical Biotechnology Vol 13. Kluwer Academic/Plenum Publishers, New York 2002, Yazdi and Murphy, (1994) Cancer Research 54, 6387-6394, Widera et al., (2003) Pharmaceutical Research 20, 1231-1238; Lee et al., (2005) Arch. Pharm. Res. 28, 722-729. Examples of formulation methods are as follows:
- Method #1 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., ⁇ 20° C. or ⁇ 80° C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl.
- Method #2 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., ⁇ 20° C. or ⁇ 80° C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl and containing 10-20 mg/L Polysorbate 80.
- Method #3 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., ⁇ 20° C. or ⁇ 80° C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0).
- Method #4 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., ⁇ 20° C. or ⁇ 80° C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0) containing 10-20 mg/L Polysorbate 80.
- Method #5 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against water, freeze dried and stored at 4° C., ⁇ 20° C. or ⁇ 80° C.
- Method #6 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against 0.01 M-0.2 M NaCl (pH 7.0-8.0), freeze dried and stored at 4° C., ⁇ 20° C. or ⁇ 80° C.
- the thio-albumin of the invention (and/or its conjugated form) may be used to produce nanoparticles and/or be entrapped within a nanoparticle or liposome.
- the thio-albumin of the invention may be used with and/or in and/or as a nanoparticle and/or liposome.
- a problem of current conjugation strategies is maintaining both the pharmacological and immunological activity of the conjugation partner, such as a bioactive-targeting ligand conjugate.
- the conjugation partner such as a bioactive-targeting ligand conjugate.
- conjugation partners There is likely to be a maximum number of protein targeting ligand/bioactive moieties (conjugation partners) possible for conjugation to a protein and if this number is exceeded the targeting ligand does not retain its biological activity.
- the biological activity of the conjugation partner is not reduced by conjugation to an albumin of the invention.
- Liposomes and nanoparticles may be used to entrap bioactive compounds. They provide a mechanism for enhanced delivery of drugs such as bioactive compounds, or uptake by target cells and/or a reduction in the toxicity of the free bioactive to non-target organs which may result in an increased therapeutic index and/or reduced side effects.
- drugs such as bioactive compounds
- many solvent-based formulations required for the delivery of some bioactive compounds are associated with toxicity which limits the maximum dose which can be given to a patient.
- Liposome and nanoparticle delivery may also be advantageous for such bioactive compounds, since they would allow larger amounts of the bioactive compound to be delivered whilst avoiding some of the toxicities of solvent-based formulations (Hawkins et al (2008) Advanced Drug Delivery Reviews, 60, 8, p 876-885).
- Attachment methods may be non-covalent or covalent. Covalent reactions appear to be favourable, because covalent linkage is more stable than noncovalent methods.
- Lipids for the covalent or non-covalent attachment of proteins, peptides, or drugs to the liposome surface are available commercially (for example Avanti Polar Lipids Inc Alabaster, Ala., USA). There are 3 major classes of functionality: conjugation through disulphide or thioether formation, amide bond formation, or biotin/streptavidin binding, any of these may be used in the invention.
- Functionalized lipid anchors commonly added to liposomes include, but are not limited those containing maleimide such as N-[4-(p-maleimidophenyl) butyramide]-PE (N-MPB]-PE) or N-[4-(p-maleimidomethyl) cyclohexane-carboxamide) (MCC-PE) which allow convenient covalent coupling of the targeting moiety via a stable thioether bond (Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288).
- maleimide such as N-[4-(p-maleimidophenyl) butyramide]-PE (N-MPB]-PE) or N-[4-(p-maleimidomethyl) cyclohexane-carboxamide) (MCC-PE) which allow convenient covalent coupling of the targeting moiety via a stable thioether bond (Martin & Papahadjopoulos (1982) J. Biol. Chem
- Method #7 Following purification the free thiol containing albumin mutein of the invention or the conjugate can be formulated into nanoparticles prepared according to known procedures for preparing nanoparticles, such as procedures disclosed in WO 2004/071536 A1 and WO 2008/007146 A1, both incorporated herein by reference.
- materials for the formation of nanoparticles including but are limited to Poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and COOH-PLA are commercially available and may be functionalized with maleimide or other known chemistries according to known literature for nanoparticle formation. Any of these may be used in or with the invention.
- PLA Poly(lactic acid)
- PLGA poly(lactic-co-glycolic acid)
- COOH-PLA are commercially available and may be functionalized with maleimide or other known chemistries according to known literature for nanoparticle formation. Any of these may be used in or with the invention.
- Another convenient way for covalent coupling of ligands to liposomes involves conjugation of two thiols to form a disulphide; however under the reductive conditions in serum more stable conjugation chemistries involving one free thiol group may be preferred.
- Chemistries such as (PDP-PE) allow covalent coupling via a disulphide bond.
- Modification of the ligand to introduce a free thiol group or a functionalized linker may be used.
- An advantage of the thio-albumin of the invention is that no ligand modification is required. However, ligand modification may optionally be used in addition to the invention.
- thiol groups are not present in proteins, or are not present in sufficient amounts or at the desired location.
- heterobifunctional cross linking agents described herein with reference to conjugation.
- Some heterobifunctional cross linking agents such as SPDP and SATA require a de-protection step.
- the thio-albumin of the invention overcomes the requirement for this additional processing.
- thio-albumin could be conjugated to liposomes or nanoparticles by other chemistries, known to the art.
- thio-albumin could be attached by an amide bond using a functionalised lipid anchor with either amine or carboxyl functional groups (examples include DSPE-PEG-COOH) which reacts with the primary amine of the ligand.
- a functionalised lipid anchor with either amine or carboxyl functional groups (examples include DSPE-PEG-COOH) which reacts with the primary amine of the ligand.
- Direct cross linking between primary amines and the surface of liposomes may also be used.
- the one or more free thiol groups of thio-albumin would then be available for conjugation to another conjugation partner.
- a conjugation partner e.g. bioactive molecule
- a conjugation partner may show a reduction in its activity (e.g. bioactivity).
- Thio-albumin described in this invention may overcome this problem by providing a conjugate, nanoparticle and/or liposome in which the conjugation partner is located and/or orientated with respect to a thio-albumin such that the conjugation partner retains at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of its unconjugated activity.
- conjugation partner includes bioactive agents, imaging agents, diagnostic agents, contrast agents and therapeutic compounds such as chemotherapeutic drugs and radiopharmaceuticals.
- a thio-albumin of the invention may be conjugated to one or more conjugation partners.
- a diagnostic agent is any pharmaceutical product used as part of a diagnostic test (i.e. together with the equipment and procedures that are needed to assess the test result).
- the diagnostic agent may be used in vivo, ex vivo or in vitro.
- a Gadolinium-DTPA-albumin conjugate may be used as a combined diagnostic and therapeutic tool to visualize and monitor, for example, dystrophic muscle by magnetic resonance imaging (MRI) and for the delivery of putative therapeutics bound to albumin for effective targeting to dystrophic muscle (Amthor et al. (2004) Neuromuscular Disorders 14912: 791-796). Malignant tumours often show an increased uptake and metabolism of albumin.
- MRI magnetic resonance imaging
- the use of gadolinium-albumin conjugate has also been described for improved imaging of malignant tumours and to determine by MRI tumours sensitive to a therapy with drug-conjugated albumin (Kiessling et al. (2002) Investigative Radiology 37(4): 93-198).
- albumin conjugates may be especially useful to increase the half life of imaging agents and would therefore permit imaging over an extended period of time.
- WO2005/082423 describes the use of serum albumin conjugated to fluorescent substances for imaging.
- a thio-albumin of this invention may be conjugated to two or more molecules selected from imaging agents, diagnostic agents, therapeutic compounds and contrast agents.
- Plasma samples show enhanced uptake of albumin (EPR: Enhanced Permeation and Retention).
- Albumin conjugates may be used for enhanced imaging, and also to assess whether tumours (or or other tissues and organs) would be suitable for albumin conjugated drugs.
- the bioactive compound may be a therapeutic or diagnostic compound.
- the therapeutic compound may be a chemotherapy drug for use in cancer chemotherapy. It may be cytostatic or cytotoxic; it may be a tumor-inhibiting agent.
- the bioactive compound may already contain a free thiol group, e.g. a polypeptide containing a Cysteine residue with a free thiol group.
- the bioactive compound may be modified so as to contain a free thiol group.
- the amino acid sequence of a polypeptide may be altered so as to include a Cysteine residue with a free thiol group, or the bioactive compound may be chemically derivatized to include a free thiol group.
- the bioactive compound may be a polypeptide (protein), particularly a recombinant protein pharmaceutical. It may be a chemotherapy or radiotherapy drug used to treat cancers and other related diseases.
- the free thiol containing albumin mutein of the invention can be conjugated via the free thiol group, or groups if the albumin mutein of the invention contains more than one free thiol, to at least one bioactive compound by methods know to the art.
- the bioactive compound includes but is not limited to, peptides, polypeptides or proteins (either natural, recombinant, or synthetic) (Debinski, (2002) Cancer Investigation 20, 801-809, O'Keefe and Draper et al., (1985) JBC 260, 932-937, Xia et al., (2000) J.
- nucleic acids and radionuclides including DNA, RNA (including siRNA) and their analogs (Lee at al., (2005) Arch. Pharm. Res. 28, 722-729, Huang et al., (2007) FASEB J. 21, 1117-1125, Daniels, T. R. et al. (2006) Clinical Immunology 121, 159-176 and the references included therein) and devices (Humphries, et al., (1994) J. Tissue Culture Methods 16, 239-242 and the references included therein). Additionally the entity can itself be modified by methods known to the art.
- a human C-C chemokine A human L105 chemokine, A human L105 chemokine designated huL105_3.
- Complement Component C1q C Adenoid-expressed chemokine (ADEC), aFGF;
- FGF-1 AGF, AGF Protein, albumin, an etoposide, angiostatin, Anthrax vaccine, Antibodies specific for collapsin, antistasin, Anti-TGF beta family antibodies, antithrombin APM-1; ACRP-30; Famoxin, apo-lipoprotein species, Arylsulfatase B, b57 Protein, BCMA, Beta-thromboglobulin protein (beta-TG), bFGF; FGF2, Blood
- myofibrillar protein Troponin I FSH, Galactosidase, Galectin-4, G-CSF, GDF-1, Gene therapy, Glioma-derived growth factor, glucagon, glucagon-like peptides, Glucocerebrosidase, glucose oxidase, Glucosidase, Glycodelin-A; Progesterone-associated endometrial protein, GM-CSF, gonadotropin, Granulocyte chemotactic protein-2 (GCP-2), Granulocyte-macrophage colony stimulating factor, growth hormone, Growth related oncogene-alpha (GRO-alpha), Growth related oncogene-beta (GRO-beta), Growth related oncogene-gamma (GRO-gamma), hAPO-4; TROY, hCG, Hepatitus B surface Antigen, Hepatitus B Vaccine, HER2 Receptor Mab, hirudin, HIV
- SMIP Small Modular ImmunoPharmaceuticalTM
- dAb Fab′ fragments, F(ab′)2, scAb, scFv or scFv fragment
- plasminogen Influenza Vaccine
- Inhibin alpha Inhibin beta
- insulin insulin-like growth factor
- Integrin Mab inter-alpha trypsin inhibitor
- interferon gamma-inducible protein IP-10
- interferons such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species
- interferons such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species
- Interferons such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species
- the albumin may also be fused to one or more purification tags such as (Ala-Trp-Trp-Pro) n , avidin/streptavidin/Strep-tag, BCCP, B-tag (VP7 protein region of bluetongue virus), calmodulin binding protein (CBP), cellulose binding domains (CBD's), chitin binding domain, chloramphenicol acetyltransferase, c-myc, dihydrofolate reductase (DHFR), FLAGTM peptide (DYKDDDDK), galactose-binding protein, glutathione-S-transferase (GST), green flourescent protein (GFP), Growth hormone, N-terminus, hemagglutinin influenza virus (HAI), His-patch thioredoxin, His-tag, HSB-tag, KSI, lacZ ( ⁇ -Galactosidase), maltose binding protein (MBP), NusA,
- HSA has ligand binding and esterase activities, as described in “All about Albumin”, T. Peters Jr., Academic Press N.Y.
- the ligand binding properties include binding to anionic and neutral ligands such as long-chain fatty acids, bilirubin and other miscellaneous ligands.
- the long-chain fatty acids, oleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and palmitoleic (C16:1) are known to bind HSA.
- the polypeptide may include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially reduce the useful ligand-binding, immunological or receptor binding properties of albumin, for example to FcRN, bilirubin and/or a fatty acid.
- the polypeptide may have at least 5%, 10%, 15%, 20%, 30%, 40% or 50%, 60%, 70%, at least 80%, 90%, 95%, 100%, 105% or more of human serum albumin's receptor binding activity, mole for mole.
- the polypeptide may have increased affinity for an albumin receptor.
- Ligand binding studies can be performed on HSA and thio-albumins using an isothermal titration calorimetry method that had been suitably qualified for this purpose.
- Samples can be pre-treated by defatting (Sogami, M. and J. F. Foster (1968). Biochemistry 7(6): 2172-82, incorporated herein by reference) followed by thiol blocking (Sogami, M., H. A. Petersen, et al. (1969). Biochemistry 8(1): 49-58, incorporated herein by reference) and subsequent gel permeation chromatography.
- the binding curves generated for thio-albumins and HSA with octanoate, for example, may subsequently be compared, and functional similarity established.
- the albumin mutein (thio-albumin) of the invention can be covalently linked to one or more conjugation partners such as bioactive compounds by methods known in the art (for example those provided by Pierce, Thermo Fisher Scientific, Rockford, IL, USA; http://www.piercenet.com/files/1601361Crosslink.pdf).
- thiol reactive group into or onto the conjugation partner, for example by incorporating or engineering another free thiol present on the conjugation partner; or by incorporating or engineering a pyridyl disulphide group on the conjugation partner; or by incorporating or engineering an iodoacetyl group on the bioactive compound or or by incorporating or engineering a maleimide group on the conjugation partner.
- N-ethylmaleimide (NEM, Pierce), 2-amino-2′-aminoethanethiolsulfonate (Pierce), N-beta-maleimidoprpionic acid (BMPA Pierce), methyl methane thiosulfonate (MMTS, Pierce), fluorescein-5-maleimide (Pierce), 5-iodoacetamido-fluorescein (5-IAF, Pierce) or N-[6-7-amino-4-methylcoumarin-3-acetamido) hexyl]-3′[2′-pyridyldithio] propionamide (AMCA-HPDP, Pierce).
- the conjugation partner may be cross-linked to the albumin mutein of the invention by methods known to the art such as, but not limited to, oxidation or by the use of cross-linking reagents such as, but not limited to, 1,4-Bis-maleimidibutane (BMB, Pierce); 1,4-Bis-maleimidyl-2,3-dihydroxybutane (BMDB, Pierce); Bis-maleimidohexane (BMH, Pierce), Bis-maleimidoethane (BMOE, Pierce); 1,8-Bis-Maleimidotriethyleneglycol (BM[PEO]3 Pierce); 1,11-Bis-Maleimidotetraethyleneglycol (BM[PEO]4 Pierce); 1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane (DPDPB, Pierce); dithuio-bis-maleimidoethane (BMB, Pierce); 1,4-Bis-maleimi
- the conjugation partner does not contain a thiol reactive group then it may be modified to incorporate one or more such groups by either chemical modification or genetic engineering by methods know to the art (Chapman, A.P. (2002) Adv. Drug Deliv. Rev., 54 531-545: Humphreys, D. P. et al. Protein Engineering, Design & Selection vol. 20 no. 5 pp. 227-234, 2007). While these two references describe methodologies to cross-link PEG to an engineered free thiol within an antibody or antibody fragment, the techniques may be used to cross-link a conjugation partner to an engineered free thiol within the albumin mutein of the invention. Alternatively the Drug Affinity Complex (DACTM) technology developed by ConjuChem Inc.
- DACTM Drug Affinity Complex
- each DACTM construct (Montreal, Quebec, Canada, H2X 3Y8) may be used, e.g. as described in WO200069902.
- the drug component the portion responsible for biologic activity
- a linker attached to the drug component and 3) a reactive chemistry group at the opposite end of the linker, usually a soft electrophile selective for thiols; a maleimide is the most useful embodiment.
- Other applicable conjugation methods are described in WO2007/071068 incorporated herein by reference.
- the conjugation partner does not contain a thiol reactive group but does contain one or more amino groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-5-azido-2-nitrobenzoyloxysuccinimide (AMAS, Pierce), N-[beta-maleimidopropyloxy] succinimide ester (BMPS, Pierce), N-eta-maleimidocaproic acid (EMCA, Pierce), N-[eta-maleimidocaproyloxy]succinimide ester (EMCS, Pierce), N-[eta-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS, Pierce), N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS, Pierce), N-[gamma-maleimidobutyryloxy]
- the conjugation partner does not contain a thiol reactive group but does contain one or more carbonyl (oxidised carbohydrate) groups then it can be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[eta-maleimidocaproic acid]hydrazide (EMCH, Pierce), 4-[N-maleimidomethyl]cyclohexane-1carboxylhydrazide.HCl.1/2 dioxane (M2C2H, Pierce), 3-maleimidophenyl boronic acid (MPBH, Pierce) and 3-[2-pyridyldithio]propionyl hydrazide (PDPH, Pierce).
- cross-linking reagents such as, but not limited to, N-[eta-maleimidocaproic acid]hydrazide (EMCH, Pierce), 4-[N-maleimidomethyl]cyclohexane-1
- the conjugation partner does not contain a thiol reactive group but does contain one or more hydroxyl groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[p-maleimidophenyl]isocyanate (PMPI, Pierce).
- cross-linking reagents such as, but not limited to, N-[p-maleimidophenyl]isocyanate (PMPI, Pierce).
- the conjugation competence of polypeptides of the invention may be tested by fluorescent labelling and cellular uptake, as described by McGraw et al., (1987), The Journal of Cell Biology, 105, 207-214 and Presley et al., (1993), The Journal of Cell Biology, 122, 1231-1241.
- Other methods of testing conjugation competence include conjugating the albumin to another molecule such as HRP.
- the mass of the resultant conjugate and/or the activity of the conjugated compound may be assayed, for example by mass spectrometry or by enzyme assay.
- a host strain suitable for use in the present invention includes an hsp150-deficient version of DXY1, disclosed in S. M. Kerry-Williams et al. (1998) Yeast 14:161-169.
- WO 95/33833 teaches the skilled person how to prepare hsp150-deficient yeast. This host strain may be referred to as ‘Strain 1’.
- the HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 0 073 646 and EP 0 286 424.
- Expression plasmids for albumin variants of this invention can be constructed in a similar way to pDB2244 described in WO 00/44772 or pDB2305 described in WO/2006/013859 for expression of human serum albumin from S. cerevisiae .
- Plasmid pDB2305 contains the HSA sequence codon-optimised for expression in S. cerevisiae .
- Alternative codon optimisation methods may be used for the particular host organism selected for thio-albumin production.
- Expression plasmids for albumin variants of this invention can also be constructed in a similar way to those described in WO 2005/061719 A1 for improved expression of human serum albumin from S. cerevisiae.
- Thio-albumin muteins can be made following modification of plasmid pDB2244 ( FIG. 7 ) or pDB2305 by site directed mutagenesis.
- Overlapping mutagenic oligonucleotide sequences can be used to modify the codon of the selected residue(s) to any DNA sequence which encodes a cysteine residue (TGT or TGC) using the procedures indicated by a commercially available kit (such as Stratagene's QuikchangeTM Kit).
- synthetic DNA fragments can be manufactured containing the desired modifications to the polynucleotide sequence.
- Plasmids pDB2243 and pDB2244 contain the native HSA gene.
- the expression cassette may or may not be codon optimised; methods for constructing expression plasmids containing HSA codon optimised for expression in S. cerevisiae are described in WO/2006/013859.
- the native nucleotide sequence encoding HSA is provided in SEQ ID No. 2.
- a HSA nucleotide sequence codon-optimised for expression in S. cerevisiae is provided as SEQ ID No. 3.
- Plasmid pDB2243 (6.203 kb) was digested to completion using restriction endonucleases Notl to release the 2.992 kb human serum albumin expression cassette.
- Plasmid pSAC35 is derivative of pSAC3 by Chinery and Hinchliffe (1989) Curr. Genet. 16 , 21-25, and in EP 286424. Plasmid pSAC35 (11.037 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce 14.037 kb pDB2244 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene ( FIG. 7 ). A person skilled in the art will appreciate that the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention.
- Plasmid pDB2690 (13.018 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce a 16.039 kb plasmid pDB2713 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene ( FIG. 9 ).
- the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention.
- plasmids for thio-albumin (i.e. conjugation competent albumin) variants of this invention could be made by subcloning synthesized DNA fragments into plasmid pDB2243 ( FIG. 8 ) prior to cloning into pSAC35 or pDB2690.
- a method for the construction of a thio-albumin subcloning plasmid containing one extra conjugation competent cysteine (relative to SEQ ID No. 1) is described, by way of example only, below
- the albumin DNA sequence of pDB2243 includes two Hindi II restriction endonuclease sites.
- the synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected codon to a cysteine codon, or an existing cysteine codon is deleted or modified to a codon for another amino acid.
- the coding sequence for the mature thio-albumin may be extended at the 5′ or 3′ end(s) or insertions made within the polypeptide to add novel sequence(s) coding for cysteine or polypeptides containing one or more cysteine.
- synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected cysteine codon to an alternative codon to create an unpaired cysteine.
- synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified by substitution of two codons at a specified site to a cysteine codon (the amino acid chain length is reduced).
- synthetic DNA may be modified such that the human serum albumin protein encoding sequence (e.g. SEQ ID No. 2 or SEQ ID No. 3 in relation to HSA) is modified by insertion of a cysteine codon at a specified site (the amino acid chain length is increased).
- Plasmid pDB2243 may be digested to completion with Hindlll restriction endonuclease and the fragment (approximately 4.383 kb) is recovered and dephosphorylated, the synthetic DNA containing the appropriate modification to the human serum albumin encoding sequence may then be cloned to produce the required thio-albumin subcloning plasmid.
- the thio-albumin subcloning plasmid may then be digested to produce an expression cassette, which may be cloned into a suitable expression plasmid in a similar manner to the construction of pDB2244, pDB2305 or pDB2713.
- a S. cerevisiae strain e.g. Strain 1
- pDB2244 WO 00/44772
- pDB2305 WO/2006/013859
- Yeast may be transformed using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-1, protocol 2; Ito et al, 1983, J. Bacteriol., 153, 16; Elble, 1992, Biotechniques, 13, 18). Transformants may be selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates.
- BMMD The composition of BMMD is described by Sleep et al., 2002, Yeast, 18, 403.
- Cryopreserved stocks may be prepared in 20% (w/v) trehalose from 10 mL BMMD shake flask cultures (24 hours, 30° C., 200 rpm).
- Thio-albumin variants with single amino acid changes were selected from Tables 5A, 5B and 6A. These variants were identified as the preferred mutations according to the methods described above. Details of each variant are given in FIG. 11 , which provides a Construct Reference (e.g. TA1 for rHA A2C), the name of the plasmid encoding each thio-albumin variant expression construct and flanking sequences required for in vivo recombination by gap-repair, and the number given to a cryopreserved yeast stock (the yeast stock number) producing each thio-albumin variant. Details of the mutant codons compared to SEQ ID No. 2 are also provided, as are the SEQ ID numbers for each thio-albumin variant (DNA and protein).
- the equivalent positions to a particular position in HSA may be determined from an alignment including human serum albumin (SEQ ID No. 1) such as FIGS. 2 and 3 .
- SEQ ID No. 1 human serum albumin
- the skilled person is familiar with alignments and can readily determine whether or not an amino acid in a sequence is equivalent to an amino acid in another sequence.
- the position of the amino acid in the nonhuman albumin is not necessarily the same relative to the N-terminal end of HSA.
- position 239 of HSA is an alanine residue
- the corresponding residue of the bovine sequence is serine-238.
- valine-479 of HSA corresponds to leucine-478 of sheep albumin.
- the plasmid pDB3927 FIG.
- pDB2244 was digested with restriction enzymes Swal and Hpal (both produce blunt ends) and self-ligated to form pDB3927.
- restriction enzymes Swal and Hpal both produce blunt ends
- pDB3927 was digested with restriction enzymes Swal and Hpal (both produce blunt ends) and self-ligated to form pDB3927.
- restriction enzyme sites were modified in the albumin DNA sequence (SEQ ID No. 2) of pDB3927 without modifying the protein sequence, as outlined below.
- the resultant DNA sequence is sequence ID No. 4.
- the coding sequence of HSA in pDB3964 is provided as SEQ ID No. 4.
- DNA synthesis and cloning was used to generate pDB3964 from pDB3927 (DNA2.0 Inc, USA).
- Synthetic DNA fragments were designed to alter specific amino acid codons within the albumin gene of pDB3964, or with combinations of modifications (see Example 3 below).
- DNA fragments containing these modifications were synthesised (DNA 2.0 Inc, USA) and cloned into pDB3964 to produce plasmids containing the thio-albumin sequences ( FIG. 11 ).
- These synthetic genes and flanking regions were excised with restriction enzymes BstEII and BsrBI from the plasmids named in FIG.
- the plasmid pDB3853 (not shown) was constructed from base vector pDB2690 (Ref DB88/WO2005/061719A1) and the synthetic linker described below.
- the synthetic linker was constructed from two oligonucleotides (Sigma-Genosys) annealed in distilled water using a temperature gradient from 96° C. to room temperature (1 min per 1° C.).
- pDB2690 was digested using Kpnl and Notl, and purified by gel extraction (Qiagen), before ligation of the annealed linker:
- pDB3936 was linearised with restriction enzymes Acc651 and BamHI before purification of the 9721bp fragment following separation by agarose gel electrophoresis.
- concentrations of the linearised pDB3936 and each of the BsrBI-BstEII fragments encoding the thio-albumin coding sequences was calculated and 100 ng of each use for each yeast transformation reaction.
- Saccharomyces cerevisiae strain BXP10 was used as the expression host throughout (So-low, S. P., J. Sengbusch, et al. (2005). “Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae .” Biotechnol Prog 21(2): 617-20.), although alternative expression hosts are also be suitable.
- Cryopreserved stocks of S. cerevisiae BXP10 were prepared from 10 mL YEPPD (1% w/v yeast extract, 2% w/v plant peptone, 2% w/v dextrose)) shake flask cultures (grown for 24 hours, 30° C., 200 rpm) mixed with an equal volume of 40% w/v sterile trehalose solution and dispensed in 1mL aliquots for storage at ⁇ 80° C.
- the pellet was resuspended in 3 mL TE/LiAc (10 mM Tris, 1 mM EDTA, pH7; 500 mM lithium acetate) and glycerol added to a final concentration of 15% (v/v), before storage in aliquots at ⁇ 80° C.
- S. cerevisiae BXP10 cells were transformed to leucine prototrophy using a modified lithium acetate method (Elble, R. “A simple and efficient procedure for transformation of yeasts.” Biotechniques 13.1 (1992): 18-20. Ito, H., et al. “Transformation of intact yeast cells treated with alkali cations.” J.Bacteriol. 153.1 (1983): 163-68.).
- 50 ⁇ l of thawed competent cells were aliquoted into a 48-well microtitre plate (Nunc) before the addition of DNA fragments for gap-repair, as described above. The plate was mixed by swirling of the plate while flat on a benchtop.
- Single colony transformants were picked and patched onto fresh BMMD agar plates for short term storage. These patches were grown at 30° C. and cells then inoculated into 10 mL BMMD shake flask cultures and cryopreserved as described earlier. 10 ⁇ l of yeast stock was inoculated into a 48-well plate containing 0.5 mL BMMD per well. Growth of cultures in microtitre plates was achieved in a humidity chamber which was a sealed Perspex box containing wet paper towels to provide ⁇ 100% humidity and evaporative loss below 0.25% over 5 days under growth conditions. The plates were incubated in the shaking humidity chamber (30° C., 200 rpm,) for 5 days at 30° C. The 48-well plate was centrifuged to pellet cells (2000 ⁇ g, 10 min, room temperature) and the supernatant was harvested.
- the concentration of the thio-albumin variants in the culture supernatants was determined by Gel Permeation High Pressure Liquid Chromatography (GP-HPLC). Protein concentrations were determined using a LC2010 HPLC system (Shimadzu) equipped with UV detection under Shimadzu VP7.3 client server software control. Injections of 25 ⁇ L were made onto a 7.8 mm internal diameter ⁇ 300 mm length TSK G3000SWXL column (Tosoh Bioscience), with a 6.0 mm internal diameter x 40 mm length TSK SW guard column (Tosoh Bioscience).
- GP-HPLC Gel Permeation High Pressure Liquid Chromatography
- Samples were chromatographed in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL.min ⁇ 1 , with a run time of 15 minutes. Samples were quantified by UV detection at 280 nm, by peak height, relative to a recombinant human albumin standard of known concentration (10 mg/mL).
- a non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of the thio-albumin variants with single mutations are compared against controls in FIG. 15 . It is evident that all of the thio-albumin variants have been successfully secreted from S. cerevisiae BXP10. Preferred mutations have high expression titres and show a sharp Coomassie stained band equivalent to rHA controls by non-reducing SDS-PAGE analysis.
- FIG. 16 describes an additional selection of thio-albumin variants with two or more free-thiol groups. Mutations shown to be expressed in Example 2 above were combined to generate sequences designed to have multiple free-thiol groups available for conjugation.
- This selection includes thio-albumin variants designed to have up to five free-thiol groups, thio-albumin variants designed to have free-thiol groups from within one Selection Group or from more than one Selection Group, thio-albumin variants designed to have free-thiol groups with and without the naturally occurring free-thiol at C34 of HSA, thio-albumin variants designed to have free-thiol groups from a range of Proximity Groups, and thio-albumin variants designed to have free-thiol groups derived from insertions, extensions, additions and/or deletions.
- FIG. 17 shows a non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of these additional thio-albumin variants compared against an rHA control (pDB3927 coding sequence).
- Cells were transferred from the shake flask to the fermenter (10 L working volume, Sartorius Biostat C 10-3 fermenter) when the concentration of cells in the shake flask has reached 0.8-1.2 g/L achieving a cell inocula concentration of ⁇ 10 mg/L (greater than or equal to 10 mg/L) in the fermenter.
- the thio-albumin variants proteins were produced by axenic culture of each of the five yeast strains in high cell density (HCD) fed-batch fermentation.
- HCD high cell density
- the aim of the fermentation was to achieve maximum biomass and productivity by controlling feed rate addition so that formation of byproducts such as ethanol and acetate were avoided. Further details of the fermentation process are described in WO96/37515.
- the temperature and pH were controlled at 30° C. and pH5.5 respectively.
- Culture supernatant was harvested by centrifugation using a Sorvall RC 3C centrifuge (DuPont) and frozen for storage, before being thawed for subsequent purification.
- FIG. 18 provides the yields of each thio-albumin variant (in g/L culture supernatant) and shows that high product titres of greater that 1 g/L culture supernatant were obtained in all cases.
- a single step chromatography procedure was used to prepare material suitable for mass spectrometry.
- This purification step used a column (bed volume approximately 200 ⁇ L) packed with AlbuPureTM matrix (ProMetic BioSciences Ltd, Cambridge UK or Novozymes Biophama UK Ltd.). This was equilibrated with 50 mM sodium phosphate, pH5.3, and loaded with neat culture supernatants, at approximately pH5.5-6.5, to approximately 40 mg protein/mL matrix. The column was washed with approximately 3 column volumes each of 50 mM sodium phosphate, pH5.3, and 50 mM ammonium acetate, pH8.0, respectively.
- Bound protein was eluted using approximately 5 column volumes of 50 mM ammonium acetate, 10 mM octanoate, pH7.0.
- the flow rate for the load step was 137 ⁇ L/min, while the wash and elution steps were performed by means of centrifugal force, using a Heraeus Multifuge 3 centrifuge at 300 rpm. Final concentrations were in the range 1.8-4.0 mg/mL and samples were approximately 2 mL volume. Free thiol determination was performed immediately after sample elution by following the procedure described below.
- the number of free thiols on a protein can be determined spectrophotometrically using Ellman's reagent.
- Ellman's reagent (5′5′-dithio-bis(2-nitronenzoic acid) (DTNB)) is an aromatic disulphide which reacts with thiol groups to form a mixed disulphide of the protein and one mole of 5-thio-2-nitrobenzoic acid (TNB) (per mole of protein sulfhydryl group). This reaction also results in a yellow colour from free TNB being released in solution.
- the number of free thiols on a protein can be determined using mass spectrometric analysis of protein sample treated with DTNB reagent.
- NTB 5-thio-2-nitrobenzoic acid
- TNB labelled and unlabelled samples were prepared for mass spectrometric analysis by desalt-ing/concentrating using Solid Phase Extaction (SPE).
- SPE columns were prepared by first wetting with 1 mL of 70% Acetonitrile (ACN Fisher)/0.1% TFA and then equilibrating ready for loading with 0.1% TFA. 1 mL of sample was loaded on the equilibrated SPE columns allowing time for the protein to bind. The bound protein and SPE columns were then washed three times in 1 mL of 0.1% Formic acid (Merck). Finally the bound protein was eluted into pre-washed 1 mL microfuge tube with 0. 5mL 70% ACN/0.1%FA.
- Time-of-Flight mass spectrometry 30 ⁇ L of sample was introduced into a hybrid quadru-pole time-of flight mass spectrometer (QqOaTOF, Applied Biosystems, QSTAR-XL®), equipped with an lonSprayTM source in positive ion mode, using flow injection analysis (FIA).
- the only instrument parameter that is actively tuned is the Decoupling Potential (DP), typically set to 250 V. Typically 2 minutes of sample scans are averaged.
- DP Decoupling Potential
- the TOF analyser is calibrated against protonated molecular ions of equine myoglobin (Sigma) and resolution is typically >14,000. Instrument control and data acquisition and processing were performed using AnalystTM QS v1.1 software (Applied Biosystems).
- thio-albumin variants produced at higher fermentation yields were preferred for analysis by the mass spectroscopy method described above. Therefore, the recombinant proteins rHA (A2C, L585C) (total of 3 free thiols), rHA (D129C, C360S, L585C) (total of 4 free thiols), and A2C rHA-Cys (total of 3 free thiols) were analysed by ESI TOF (electrospray ionsation time of flight) mass spectrometry pre- and post-DTNB treatment to determine the numbers of free thiols present on each molecule.
- ESI TOF electrospray ionsation time of flight
- the rHA (A2C, L558C) thio-albumin variant is particularly surprising in that it provides more than the expected number of reactive groups available for conjugation. Also present is a series of peaks ⁇ 396 Da apart which are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA (A2C, L558C) molecule. This adduct formation is known to occur in the presence of excess DTNB.
- the rHA (D129C, C360S, L585C) thio-albumin variant is particularly surprising in that it has provided more than the expected number of groups available for conjugation. Also present are a series of peaks ⁇ 396 Da apart these are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA D129C, C360S, L585C. This adduct formation is known to occur in the presence of excess DTNB.
- conjugation competent cysteines can be in regions of that may or may not have secondary structure, and/or may or may not be generated from natural disulphide bonds, and/or may or may not be additional cysteines residues (such as cysteine residues extending from the natural C-terminus of HSA).
- TA35 i.e. A2C, A364, D562 in addition to naturally occurring C34
- TA33 i.e. A2C, L585C in addition to naturally occurring C34
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Evolutionary Biology (AREA)
- Theoretical Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Physiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biotechnology (AREA)
- Medical Informatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
- This application is a division of U.S. patent application Ser. No. 13/201,123 filed Aug. 11, 2011, pending, which is a 35 U.S.C. 371 national application of PCT/EP2010/051751 filed Feb. 11, 2010, which claims the benefit of priority to European application nos. 09152625.1 and 09152686.3, filed Feb. 11, 2009 and Feb. 12, 2009, respectively, and U.S. provisional application No. 61/154,555, filed Feb. 23, 2009. The contents of each of U.S. patent application Ser. No. 13/201,123, PCT application no. PCT/EP10/51751, European application no. 09152625.1, European application no. 09152686.3, and U.S. provisional application No. 61/154,555 are fully incorporated herein by reference.
- This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
- The present invention relates to conjugation competent albumins and albumin-related polypeptides, and their conjugates with at least one moiety, and to polynucleotides encoding them.
- Serum albumins provide valuable scaffolds to which bioactive molecules may be fused, either through genetic fusions or chemical fusions to improve the properties of the fused molecule(s) (Leger, R. et al. (2004). Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau, K., et al. (2005). Bioconjug Chem 16(4): 1000-8; Balan, V. et al. (2006). Antivir Ther 11(1): 35-45;
EP 0 413 622; WO 90/13653;EP 1 681 304; WO 1997/024445; WO 01/79271). Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused bioactive molecule from inactivation. - The biochemistry, genetics and medical applications of albumins are well characterized (“All about Albumin”, T. Peters Jr., Academic Press N.Y., and http://www.albumin.org/). Human serum albumin (HSA, also referred to as HA) is the most abundant protein in human plasma at ˜60 g/L. The sequence of HSA is provided in SEQ ID No. 1. Natural variants of HSA occur and a list of know polymorphisms is given in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and at http://www.uniprot.org/uniprot/P02768.
- The production and purification of recombinant human albumins are well established (
WO 95/23857;WO 00/44772; WO 2006/066595;EP 0 305 216; Sleep et al. 1990 Biotechnology (NY). 1990 January; 8(1):42-6)) and include recombinant human albumin for pharmaceutical applications (Bosse et al. (2005). J Clin Pharmacol 45(1): 57-67). The three-dimensional structure of HSA has been elucidated by X-ray crystallography (Carter et al. (1989). Science 244(4909): 1195-8; Sugio et al. (1999). Protein Eng 12(6): 439-46). The HSA polypeptide chain has 35 cysteine residues, which form 17 disulphide bonds and one unpaired (free) cysteine atposition 34 of the mature protein (Seq ID No. 1). Cysteine-34 has been used to for conjugation of molecules to albumin (Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8), and provides a precise, well defined site for conjugation. However, conjugation at cysteine-34 provides only one site for attachment of a single moiety thus there is no choice of conjugation site. Also, the provision of a single conjugation sites means that only one moiety can be conjugated to each albumin molecule. What is required is an albumin molecule which provides one or more alternative attachment sites. - Based on an analysis of the three-dimensional structure of a human serum albumin (HSA), conserved residues within albumin polypeptides and natural polymorphisms thereof, the inventors have designed variant polypeptides (muteins) of albumin which have one or more conjugation competent cysteine residues. The term ‘thio-albumin’ is used herein to describe an albumin variant which comprises one or more unpaired cysteine residues, particularly an albumin variant in which one or more of the unpaired cysteine residues does not occur in a naturally occurring variant of an albumin. Thus a thio-albumin is a ‘conjugation competent albumin’. A thio-albumin may be referred to as a ‘cysteine variant of an albumin’.
- Throughout this specification, the term ‘albumin’ includes naturally occurring albumin, albumin-related proteins and variants thereof such as natural and engineered variants. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins. The albumin may have at least 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99% similarity or identity to SEQ ID No. 1. Thus a thio-albumin of the invention may be a derivative of, or be based on, any of such albumin.
- The unpaired cysteine residues may be provided by insertion, deletion, substitution, addition or extension of an albumin sequence.
- The invention also relates to a conjugate comprising at least one, for example 2, 3, 4, 5 or 6, conjugation partners such as bioactive compounds and a polypeptide according to the invention
- The invention also provides a method for designing conjugation-competent albumins.
-
FIG. 1 . is a table showing criteria used to select sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of conjugation competent cysteines. -
FIG. 2 . is an alignment of the amino acid sequence of human serum albumin (SEQ ID No. 1=Human-P02768.pro) with albumins from fifteen other mammalian species. ‘Majority’ shows the consensus sequence. ‘+ Majority’ shows the relative homology between all sixteen sequences in bar chart form, where the height of the bar indicates the relative homology at 20, 40, 60, 80 and 100%. The protein sequences include the leader sequence. -
FIG. 3 . Is an alignment of the amino acid sequence of human serum albumin (P02768.pro=SEQ ID No. 1) with albumins from thirty two other species, some of which are mammalian. ‘Majority’ shows the consensus sequence. ‘+ Majority’ shows the relative homology between all thirty three sequences in bar chart form, where the height of the bar indicates the relative homology at 20, 40, 60, 80 and 100%. The protein sequences include the leader sequence. P02768.pro: human; P02769.pro: bovine; P49064.pro: cat; P49822.pro: dog; Q5XLE4.pro: donkey; JC5838.pro: gerbil; ACF10391,1.pro: goat fragment; AAQ20088.pro: guinea pig; P35747.pro: horse; Q28522.pro: macaque; P07724.pro: mouse; P08835.pro: pig; P02770.pro: rat; P49065.pro: rabbit; Q28522.pro: Rhesus monkey; P14639.pro: sheep; NP_001127106.pro: orangutan; P19121.pro: chicken; P01012.pro: chicken ovalbumin; O73860.pro: turkey ovalbumin; AAC63407.pro: sea lamprey; Q91274.pro: sea lamprey; P21847.pro: bullfrog; AAD09358.pro: Rana shqiperica; ABXL68.pro: Xenopus; NP_001004887.pro: Xenopus; AAL56646.pro: Spotted Salamander; Q03156.pro: Atlantic salmon; P21848.pro: Atlantic salmon; AAM46104.pro: Sphenodon punctatus; P83517.pro: Australian lungfish; S59517.pro: monocled cobra; AAL08579.pro: Schistosoma mansoni. -
FIG. 4 . is a Venn diagram showing the classes of and relationship between twenty amino acids. -
FIGS. 5A, 5B, 5C and 5D are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of one or more conjugation competent cysteine. -
FIGS. 6A and 6B are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for disruption of one or more disulphide bonds for the generation of one or more conjugation competent cysteines. -
FIG. 7 . is a map of plasmid pDB2244. -
FIG. 8 is a map of plasmid pDB2243. -
FIG. 9 . is a map of plasmid pDB2713. -
FIG. 10 is a table showing preferred sites for conjugation grouped according to their relative position on a folded albumin of SEQ ID No. 1. -
FIG. 11 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having a single free thiol group in addition to Cys-34 of native human serum albumin. -
FIG. 12 is a map of plasmid pDB3927. -
FIG. 13 is a map of plasmid pDB3964. -
FIG. 14 is a map of plasmid pBD3936. -
FIG. 15 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation) of albumin molecules having a free thiol group at Cys-34 of SEQ ID No. 1 and an additional free-thiol at the position indicated below the bar chart. -
FIG. 16 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed. -
FIG. 17 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation of albumin molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed. -
FIG. 18 is a table showing the fermentation yield and relative level of conjugation to albumin molecules comprising one or more free-thiols. -
FIG. 19 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) before treatment with DTNB. -
FIG. 20 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) after treatment with DTNB. -
FIG. 21 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) before treatment with DTNB. -
FIG. 22 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) after treatment with DTNB. -
FIG. 23 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol) before treatment with DTNB. -
FIG. 24 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol). - A first aspect of the invention provides a method for designing and/or preparing variant albumins comprising one or more conjugation competent cysteine residues. Therefore, the polypeptide may be considered to be conjugation-competent. Such an albumin may be referred to as a ‘thio-albumin’ or as a ‘cysteine varant’ of an albumin. The term ‘conjugation competent cysteine’ includes a cysteine which has a thiol which is not disulphide bonded to another cysteine and which is, preferably, not blocked from conjugating to another molecule (which may be referred to as a ‘conjugation partner’) due to unfavourable steric hindrances. That is, preferably the location of the cysteine within or on a folded polypeptide is such that it is available for conjugation.
- A number of selection criteria may or may not be used alone or in any combination in order to identify suitable sites for introduction of a conjugation competent cysteine residue. Therefore, the invention provides a method and/or rules for a priori identification of sites of an amino acid sequence of albumin at which a conjugation competent cysteine may be introduced. Such sites may be referred to as ‘candidate residues’. The albumin sequence on which the variant albumin is based may be SEQ ID No. 1 or any other albumin. For example, the variant albumin may be be based on an albumin which does or does not have a cysteine at
position 34 of the amino acid sequence, or an equivalent position. Cysteine residues may or may not be introduced by one or more of substitution, insertion, deletion, extension and addition. Sites may or may not be selected with reference to a 3-dimensional structure of an albumin or variant thereof. The following criteria may or may not be used to select suitable sites: - (a) Solvent Accessible Surface Area (“Surface Accessibility” (% SASA)).
- Preferably the surface accessibility is high. For example, preferably the surface accessibility is at least 60%, more preferably, from 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% to 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%. % SASA may be determined as a ‘raw score’ using the methods described herein or may be calculated relative to the score of the residue which has the maximum surface accessibility in the protein. For example, the albumin of HSA 1AO6 has a maximum surface accessibility of 229.0 and this is the highest scoring residue in HSA. A higher surface accessibility indicates that the residue is on the surface of the protein and is therefore available for binding. Such accessibility may be calculated using a method as described herein.
- (b) Presence or Absence of Crystallographic B-Factor(s).
- B-factor indicates relative flexibility of an amino acid residue within a 3-dimensional structure. Preferably the B-factor is from at least 30, 40, 50, 60, 70, 80 or 90% to at least 40, 50, 60, 70, 80, 90 or 100% which may or may not be relative to the maximal B-factor score of any amino acid residue within the molecule. For HSA (e.g. 1AO6), preferably the B-factor score is high, for example from at least 30, 40, 50, 60, 70, 80, 90, or 100 to at least 40, 50, 60, 70, 80, 90, 100 or 106 (for example using the B-factor scoring system described herein). Alternatively the B-factor score may be less than or equal to 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10%, as described herein.
- The B-Factor (root mean square fluctuations) of the C-alpha carbon atoms during the last nanosecond of the simulation may be calculated using the Gromacs tool “g_rmsf”, version 3.3, based on D. van der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark and H. J. C. Berendsen: GROMACS: Fast, Flexible and Free, J. Comp. Chem. 26 pp. 1701-1718 (2005).
- (c) Presence of Absence of Secondary Structure (SS).
- The candidate residue may or may not be located within secondary structure for example H (Helix), B (isolated beta bridge) or E (Extended sheet). Location of the residue outside of secondary structure indicates that the residue is less likely to be important to secondary structure and/or is more likely to be available for binding than a residue located within secondary structure.
- (d) Relative Homology with Other Albumins.
- Within a given protein sequence, an amino acid residue showing high homology with other similar sequences is likely to indicate such a residue or region is likely to be important to the structure and/or function of the protein. Therefore it is preferred that a candidate residue shows a homology of less than 100% relative to alignment of the albumin in which the residue is located with known albumins (e.g. mammalian albumins such as those shown in
FIG. 2 or a combination of mammalian and non-mammalian albumins such as those shown inFIG. 3 ). A homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 is preferred. Homology can be determined using algorithms known in the art such as Clustal, e.g. Clustal W (Thompson et al. (1994). Nucleic Acids Res 22(22): 4673-80) or Clustal V (Higgins, D. G. and P. M. Sharp (1989). “Fast and sensitive multiple sequence alignments on a microcomputer.” Comput Appl Biosci 5(2): 151-3.). Lower homology indicates that the residue is not particularly important or critical to the structure and/or function of the protein. Preferably the homology is determined with reference to the sixteen mammalian albumins ofFIG. 2 or the thirty three mammalian and non-mammalian albumins ofFIG. 3 . - (e) Presence or Absence of Adjacent Conserved Residues.
- Within an amino acid sequence, each residue has one or two adjacent residues. If a candidate residue is immediately adjacent one or more residues having a low homology, relative to known albumins, this indicates that the candidate residue is unlikely to be particularly important or critical to the structure and/or function of the protein. This is because the candidate residue is likely to be located within a relatively unconserved region of the protein. It is therefore preferred that the candidate residue is not adjacent a residue which has 100% homology relative to alignment of the albumin with known albumins. Homology may be determined as described herein. The candidate residue may be adjacent two residues (i.e. one residue C-terminal relative to the candidate residue and one residue N-terminal relative to the candidate residue) which each have 100% homology relative to alignment of the albumin with known albumins (e.g.
FIG. 2 orFIG. 3 ). It is preferred that the candidate residue is adjacent one or two residues having a homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5—these levels of homology may be referred to as ‘thresholds’. Homology may be determined as described herein. Taking into account the homology threshold, the location of an amino acid relative to a conserved region may be quantified, for example by scoring an amino acid which is not adjacent any amino acid exceeding the homology threshold as 0, scoring an amino acid which is adjacent one amino acid exceeding the threshold as 1 and scoring an amino acid which adjacent two amino acids exceeding the threshold as 2. - (f) Evidence for Polymorphism(s).
- A polymorphism is a genetic variation, a polymorphism may or may not cause a phenotypic change to the resultant protein. Preferably the candidate residue is not at a position for which a polymorphism causing a phenotypic change is known. More preferably, the candidate residue is not at a position for which a polymorphism causes, or is known to cause, thermal instability. Polymorphisms known for HSA (SEQ ID No. 1) are detailed in
FIG. 1 and are also discussed in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16 and at http://www.uniprot.org/uniprot/P02768. The presence, absence and/or effect of a polymorphism may be quantified, for example by scoring a known polymorphism that has no phenotypic change as 0, scoring a polymorphism where a phenotypic change is known (but not known to cause thermal instability) as 1 and scoring a polymorphism which is known to cause thermal instability as 2. - (g) Relative Conservation of Candidate Amino Acid and Cysteine.
- Amino acids fall into various well known classes. Therefore, some amino acids are more closely related than others. The introduced cysteine residue may or may not maintain a relatively high level of conservation with the candidate amino acid.
FIG. 4 is a Venn diagram which provides one system by which conservation level can be quantified. The scoring system ofFIG. 4 uses a scale of 0 to 5 in which substitutions of high conservation have a score of 0, substitutions of low conservation have a score of 5 and substitutions of intermediate conservation have a score of 1, 2, 3 or 4. Preferably substitution of the candidate residue is not an unconserved substitution, that is preferably (using the scoring system ofFIG. 4 ) the candidate residue does not have conservation score (relative to cysteine) of 5. More preferably the candidate residue has a higher conservation relative to cysteine (e.g. a score of 4, 3, 2 and, more preferably, 1). The scoring system is described in the section entitled ‘Conservative Substitution’ (below). - (h) Expression Level.
- The thio-albumin may or may not be capable of being expressed at a level of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to the expression of an unmodified albumin (such as SEQ ID No. 1) from a suitable expression system, such as yeast (e.g. Saccharomyces, e.g. S. cerevisiae) or an Aspergillus. Relative expression levels can be determined, for example, by expression of the protein followed by quantification by SDS-PAGE, HPLC or Western Blotting.
- (i) Conjugation Competence.
- The thio-albumin may or may not have a high level of conjugation competence, for example at least 50, 60, 70, 80, 90, 95 or 100% relative to the conjugation competence of an albumin consisting of SEQ ID No. 1 having only one conjugation competent cysteine at Cys-34. Conjugation competence may be determined relative to any conjugatable molecule (conjugation partner) of interest, for example a bioactive molecule or a fluorescent dye. Determination may be through mass spectrometry analysis or quantification of the activity of the bioactive compound such as its fluorescence. An advantage of a thio-albumin having a high conjugation competence is that it may allow efficient conjugation of molecules to the thio-albumin. Conjugation competence may be measured with respect to time. Favoured thio-albumins may be (a) those which achieve maximal conjugation quickly or (b) slowly.
- (j) Activity of Conjugated Compound.
- The thio-albumin of the invention may be conjugated to a compound (conjugation partner), for example a bioactive compound, such that the compound has a high level of activity relative to its activity in an unconjugated state. Preferably, the conjugated compound shows at least 1, 10, 20, 40, 50, 60, 70, 80, 80 and most preferably 100% of its activity relative to its unconjugated state. An advantage of a conjugated compound with a high level of activity is that it reduces the quantity of conjugated compound required to achieve a desire effect, e.g. a desired therapeutic effect.
- (k) Receptor Binding Capacity of Albumin
- The conjugated- and/or non-conjugated thio-albumin may or may not have a receptor binding activity of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the receptor binding activity of human serum albumin (SEQ ID No. 1). Alternatively, the conjugated- and/or non-conjugated thio-albumin may or may not have a lower receptor binding activity for example at most 0, 10, 20, 30, 40, 50, 60, 70, 80 or 90% than human serum albumin. Receptor binding activity may be determined by assay, such as in relation to binding to FcRn.
-
FIG. 1 . shows the scores of each amino acid residue of HSA (SEQ ID No. 1) for each of parameters (a) to (g). For clarity, in vivo, HSA is initially produced as a 609 amino acid protein in which the first twenty four amino acids are a leader sequence. The leader sequence is cleaved off to generate a 585 amino acid mature protein. Throughout this specification, the mature protein is referred to as SEQ ID No. 1. The structure of HSA model A106 disregards the first four residues and the last three residues of SEQ ID No. 1 because these are unresolved in the 3D model. Therefore,residue 1 of model A106 is equivalent toresidue 5 of SEQ ID No. 1. Throughout this specification, all residues are cited with reference to SEQ ID No. 1, unless stated otherwise. The immature sequence of HSA (i.e. HSA with its natural C-terminal leader sequence) is provided in SEQ ID No. 102. - The column labels of
FIG. 1 . are detailed below: - Position in 1AO6: Refers to the amino acid position in the crystal structure of human serum albumin from the RCSD Protein Databank (PDB, http://www.rcsb.org/pdb/) with the entry with PDB identity 1AO6 or 1ao6 (Sugio, S., A. Kashima, et al. (1999). Protein Eng 12(6): 439-46). Note that compared to the mature HSA sequence (SEQ ID No1), the 1AO6 structure starts at residue 5S (with the first 4 amino acids absent from the structure) and finishes at 582A of SEQ ID No1 (with the last 3 amino acids absent from the structure). The amino acid positions used herein to describe positions to alter to generate conjugation competent cysteines are referring to the positions in SEQ ID No1, not 1ao6.
- Position in Mature HSA: The amino acid position in SEQ ID No. 1 taken from the 585 residue secreted form of HSA, National Center for Biotechnology Information, ACCESSION: 1AO6_A VERSION GI:3212456 (24-SEP.-2008), Chain A, Crystal Structure Of Human Serum Albumin. (Sugio et al. (1999). Protein Eng 12(6): 439-46).
- Position with Leader Sequence: Refers to the position in the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence.
- Amino Acid: The standard one letter code for each of the 20 amino acids (e.g. A=Ala=Alanine).
- % SASA: The solvent accessible surface area calculated for each residue, using the DSSP software described in Kabschand and Sander (1983). Biopolymers 22(12): 2577-637. Each solvent accessible surface area was divided by a standard value for the particular amino acid found in that position and multiplied by 100, thereby obtaining a percentage of the standard value for each residue. The standard solvent accessible surface areas for the 20 different amino acids are defined as (using one-letter codes for the amino acids): A=62, C=92, D=69, E=156, F=123, G=50, H=130, I=84, K=174, L=97, M=103, N=85, P=67, Q=127, R=211, S=64, T=80, V=81, W=126, Y=104).
- B-Factor: The crystallographic B-factor value for the C-alpha atom was extracted directly from the PDB file. The B-factor is in
column number 11 of the 1ao6 PDB file PDB, (http://www.rcsb.org/pdb/). - SS (Secondary Structure): The secondary structure determined for each residue using the DSSP software Kabsch and Sander (1983). Biopolymers 22(12): 2577-637. If the secondary structure is defined as H (Helix), B (isolated beta bridge) or E (Extended sheet), the residue is marked ‘1’, otherwise as ‘0’.
- Align 1 (Mamm. W): The homology level for an alignment of various mammalian albumin family proteins with HSA (SEQ ID NO: 1), identified as P02768 compiled using MegAlign program (DNASTAR, Lasergene, version 8.0.2) based on Clustal W; six levels of homology are determined with the highest=100%, decreasing in 20% increments, to the lowest=0% (
FIG. 2 ). - Adj. 100%'s (Align 1): The score according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with the mammalian albumins of
FIG. 2 . A score of 0 indicates the residue is not adjacent to a residue with 100% homology when HSA is aligned with the mammalian albumins ofFIG. 2 ; a score of 1 indicates that the residue is adjacent to one residue with 100% homology when HSA is aligned with mammalian albumins; a score of 2 indicates that the residue is adjacent to two residue with 100% homology when HSA is aligned with mammalian albumins. - Align 2 (Var. Sps. V): The homology level for an alignment of various albumin family proteins with HSA (SEQ ID NO: 1), identified as P02768 compiled using MegAlign program (DNASTAR, Lasergene, version 8.0.2) based on Clustal V; six levels of homology are determined with the highest=100%, decreasing in 20% increments, to the lowest=0% (
FIG. 3 ). - Polymorph: This identifies whether or not a polymorphism is known at the amino acid residue. Single amino acid polymorphisms of human serum albumin (SEQ ID NO: 1) were taken from Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768, with amino acid positions taken from the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence, and described using the standard one letter amino acid code (e.g. D25V refers to an aspartic acid being changed to a valine at
position 1 in SEQ ID NO: 1). - Phenotype Change: A score representing the ‘severity’ of phenotypic change derived from the sources of known phenotypic changes ('Polymorph.', referenced above) where; 0=no known phenotypic change, 1=a phenotypic change has been described for any of the mutations at this position compared to the residue in SEQ ID NO: 1, excluding a change resulting in decreased thermal stability, 2=a mutation at this position in SEQ ID NO: 1 is described ‘as causing reduced thermal stability.
- Conserved Mutation vs. Cysteine: A score referring to how well conserved the amino acid is compared to cysteine, as derived from
FIG. 4 (described herein), and ranging from 1 to 5 for mutations to cysteine. A score of 1 is assigned to the most conservative changes possible (e.g. alanine to cysteine), and ranging to a score of 5 for the lowest of conservation compared to a cysteine (e.g. histidine to cysteine). - Although the selection criteria can be used in any desired combination, four preferred groups of selection criteria (A, B, C, D) are described, by way of example only, below. Of these (A) and (B) may also be referred to as
Selection Groups 1 and 2 (respectively): - (A) A particularly preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence which corresponds to the first, second, third, fourth or fifth residue relative to the N- or C-terminus of the albumin sequence (of the model or of the amino acid sequence) or which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility (SASA) of at least 90%; B-factor score of at least 60; less than 80% homology to known mammalian albumins (e.g.
FIG. 2 ); no adjacent residues with 100% homology to known mammalian albumins (e.g.FIG. 2 ); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above; - substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,
- optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and
- optionally preparing a polypeptide having the required amino acid sequence.
- With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (A) include L585, D1, A2, D562, A364, A504, E505, T79 and E86 (in descending order of solvent accessibility) and are also shown in
FIG. 5A . - (B) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 90%; B-factor score of at least 40; less than 80% homology to known mammalian albumins (e.g.
FIG. 2 ); no adjacent residues with 100% homology to known mammalian albumins (e.g.FIG. 2 ); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above; - substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,
- optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and
- optionally preparing a polypeptide having the required amino acid sequence.
- With reference to model 1A06 and SEQ ID No. 1, candidate residues identified by selection criteria (B) include D129, D549, A581, D121, E82, S270, Q397 and A578 (in descending order of solvent accessibility) and are also shown in
FIG. 5B . - (C) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is ‘full length’, i.e. the mature amino acid sequence of the albumin);
- selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 50; less than 100% homology to known mammalian albumins (e.g.
FIG. 2 ); less than 80% homology to the various albumins aligned inFIG. 3 ; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above; - substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue;
- optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and
- optionally preparing a polypeptide having the required amino acid sequence.
- With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (C) are shown in
FIG. 5C . - (D) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:
- providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model);
- selecting a candidate amino acid residue in the albumin sequence which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 30; less than 100% homology to known mammalian albumins (e.g.
FIG. 2 ); less than 80% homology to the various albumins aligned inFIG. 3 ; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above; - substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,
- optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and
- optionally preparing a polypeptide having the required amino acid sequence.
- With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (D) are shown in
FIG. 5D . - Since
FIGS. 5A, 5B, 5C and 5D are selections fromFIG. 1 , the column headings are the same. - A candidate residue may be one or more of the cysteine residues involved in disulphide bonding present in the albumin molecule (in the case of HSA, SEQ ID No. 1, there are 17 disulphide bonds and therefore 34 cysteines involved in disulphide bonding). Two cysteines which are linked by a disulphide bond may be referred to as ‘counterparts’. In order to generate a conjugation competent cysteine, the candidate residue may be deleted or may be substituted with a different amino acid, particularly Ser, Thr, Val or Ala in order to create a free thiol at the partner cysteine. The 34 cysteine residues of SEQ ID No. 1 which are involved in disulphide bonding are C53, C62, C75, C91, C90, C101, C124, C169, C168, C177, C200, C246, C245, C253, C265, C279, C278, C289, C316, C361, C360, C369, C392, C438, C437, C448, C461, C477, C476, C487, C514, C559, C558 and C567. In relation to the invention, some of these 34 candidate residues are more favoured than others.
- Cysteine residues were visually inspected using the PyMOL software (Warren L. DeLano “The PyMOL Molecular Graphics System.” DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org), and the cysteines in the disulphide bonds were divided into 3 categories:
-
- those that can be replaced, for example, by serine, leaving its counterpart cysteine as a free thiol that has a high probability of being a conjugation site. These correspond to C75, C91, C124, C168, C169, C316, C360, C361, C567, C558.
- those that can be replaced by serine, leaving its counterpart as a free thiol that has a medium probability of being a conjugation site. These correspond to C90, C101, C177, C265, C279, C278, C289, C369, C392, C438, C476, C487, C514, C559.
- those that can be replaced by serine, leaving its counterpart as a free thiol that has a low probability of being a conjugation site. These correspond to C53, C62, C200, C246, C245, C253, C437, C448, C461, C477.
- The judgment is based on surface accessibility and the orientation of the C-alpha -C-beta bond of the potential free thiol relative to the folded polypeptide. Using this judgment each of the cysteine residues of HSA were given a modification score and ranked as high, medium or low.
-
FIG. 6A , provides a list of all the cysteine residues which have a high modification score (right hand column), indicating that modification of a cysteine residue at this position would result in its counterpart cysteine providing a free thiol that has a high probability of being suitable for use as a conjugation site. -
FIG. 6B , provides a list of the counterpart cysteines that that, when unpaired (thus providing a free thiol), have a high probability of being suitable for use as a conjugation site - The column labels for
FIG. 6 are the same for those described forFIG. 1 with the addition of: - Modification Score: defined as ‘high’, ‘medium’ or low' as described herein.
- Disulphide Information: summarises disulphide pairing in SEQ ID No. 1.
- (Polymorp.) Phenotype Change: summarises the columns labelled ‘Polymorph.’ And ‘Phenotype Change’ in
FIG. 1 . - Preferred cysteine residues for modification could be further selected based on the other information provided in
FIG. 6A , such as assigned secondary structure, cysteine residues with no adjacent conserved residues (100% amongst mammalian albumins (aligned by Clustal W), no known polymorphisms causing phenotypic changes. - Alternatively cysteine residues for modification could be selected by examining the environment of the cysteine residue (containing a free thiol group) generated by modification of its counterpart cysteine residue provided in
FIG. 6A , characteristics such as high % SASA may be preferred (FIG. 6B , fifth column). - For clarity, for albumins other than the human serum albumin of SEQ ID No. 1, equivalent residues are favoured for mutation. Such equivalent residues may or may not have identical residue numbers to those of SEQ ID No. 1 but would be clearly identifiable by the skilled person, for example with reference to homology alignments with SEQ ID No. 1 and other albumins such as those of
FIG. 2 and/orFIG. 3 . For example, inFIG. 2 , the residue at positions at 160 of the horizontal ‘ruler’ are equivalent but have differing residue numbers and, sometimes, are differing amino acids, e.g. L159 in human, W134 in goat fragment, L151 in Macacque and M159 in mouse. It is preferred that, for an alignment such asFIG. 2 orFIG. 3 , equivalent residues are within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid of the candidate amino acid of SEQ ID No. 1. The ‘ruler’ above the alignment indicates whether an amino acid is within 1 to 10 amino acids of a candidate amino acid of SEQ ID No. 1. - The selection criteria of Group (A) are more preferable than those of Group (B) which in turn are more preferable than those of Group (C) and in turn are more preferable than those of Group (D).
- The method may or may not further comprise determining the receptor binding capacity and/or the conjugation competence of the polypeptide and optionally selecting a polypeptide which does or does not have a receptor binding capacity and/or conjugation competence.
- ‘Preparing’ a polypeptide may or may not include expressing the polypeptide in a host cell and/or purifying the polypeptide from the host or host cell media. The method may comprise favouring selection of residues meeting one or all of the following criteria:
-
- residues having high surface accessibility are preferred to those having low surface accessibility;
- conservative mutations from another amino acid to cysteine are preferred over non-conservative mutations;
- Alternatively, or in addition, selection criteria as detailed throughout this specification may or may not be used to select residues in the method of the first aspect of the invention.
- A second aspect of the invention provides a thio-albumin comprising a polypeptide sequence and/or polypeptide designed and/or produced according to the first aspect of the invention.
- Preferably the polypeptide is a recombinant polypeptide. Preferably the polypeptide is an isolated and/or purified polypeptide. Preferably the polypeptide is synthetic and/or does not naturally occur in nature.
- Specifically, the invention provides a polypeptide which has an amino acid sequence which is at least 60% identical to
residues 1 to 585 of SEQ ID No. 1 or a fragment or fusion thereof, in which: - (a) at a position equivalent to
position 34 of SEQ ID No. 1, there is a conjugation competent cysteine residue; and - (b) elsewhere in the polypeptide there is one or more conjugation competent cysteine residues, preferably 2 or more.
- In addition the invention provides a conjugation competent polypeptide comprising an amino acid sequence which is at least 60% identical to
residues 1 to 585 of SEQ ID No. 1, or a fragment or fusion thereof, in which: - (a) at a position equivalent to
position 34 of SEQ ID No. 1, there is not a conjugation competent cysteine residue; and - (b) elsewhere in the polypeptide there is one or more conjugation competent cysteine residues, preferably 2 or more or 3 or more.
- The polypeptide may or may not comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conjugation competent cysteine residues.
- More specifically, the polypeptide (which may be described in relation to a known albumin sequence such as SEQ ID No. 1) may or may not comprise one or more of:
- (a) substitution of a non-cysteine amino acid with a cysteine at a position corresponding to a position equivalent to any of residues L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397 and A578 of SEQ ID No. 1;
- (b) insertion of a cysteine at a position adjacent the N- or C-side of an amino acid which may or may not correspond to a position equivalent to any of residues L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397 and A578 of SEQ ID No. 1;
- (c) a cysteine with a free thiol group at a position which may or may not correspond to any of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 which may or may not be generated by deletion or substitution of C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567.
- (d) addition of a cysteine to the N-side of the N-terminal residue of an albumin sequence and/or to the C-side of the C-terminal residue of an albumin sequence such that the net result of the substitution, deletion, addition or insertion events of (a), (b), (c) and (d) is that the number of conjugation competent cysteine residues of the polypeptide sequence is increased relative to the polypeptide prior to the substitution, insertion, deletion and addition events. Within (a) to (d), above, the residues all of the residues are preferred. However, within each of (a), (b), (c) and (d), the residues are listed in order of decreasing preference.
- A thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1), is modified to an amino acid which is not cysteine. For example, cysteine may or may not be replaced by an amino acid which has a relatively high conservation score (e.g. 1, 2 or 3 as calculated according to
FIG. 4 ) such as alanine or serine. A thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1) are present. - As detailed herein, the invention may be achieved by introducing cysteine residues by one or more of extension, addition, insertion, substitution or deletion.
- An addition may be made by extension and/or insertion.
- For example, one or more conjugation competent cysteines may or may not be created in an albumin by extension; e.g. by adding an extra cysteine residue to the N-terminus or C-terminus of the molecule, which may or may not be added as a single cysteine residue, or as a longer polypeptide which contains one or more conjugation competent cysteines. The cysteine residue(s) may be added immediately adjacent the N- or C-terminus of the albumin. Alternatively, there may be one or more other amino acid residues located between the N- and/or C-terminus of the albumin and the cysteine residue(s). When two or more cysteine residues are added, some or all of the added cysteines may be separated from each other by one or more other amino acids, for example by from 1 to 50 amino acids, such as from 1, 10, 20, 30, or 40 amino acids to from 10, 20, 30, 40, or 50 amino acids. A preferred N-terminal extension is the addition of Cys immediately adjacent the N-terminal of a mature albumin (i.e. albumin cleaved from its leader sequence). For example, for an albumin comprising or consisting of SEQ ID No. 1, Cys is preferably immediately N-terminal to the first Asp (D1). Such an albumin may be referred to as ‘Cys-albumin’, e.g. ‘Cys-HSA’ (where HSA is Human Serum Albumin). Other preferred N-terminal extensions of albumins such as SEQ ID No. 1 include Cys-Ala-albumin such as Cys-Ala-HSA. A preferred C-terminal extension is the addition of Cys immediately adjacent the C-terminal of an albumin, such as a mature albumin. For example, for an albumin comprising or consisting of SEQ ID No. 1, Cys is preferably immediately C-terminal to the last Leu (L585) residue. Such an albumin may be referred to as ‘albumin-Cys’, e.g. HSA-Cys. Other preferred C-terminal extensions of albumins such as SEQ ID No. 1 include albumin-Ala-Cys, such as HSA-Ala-Cys. Polypeptides suitable for providing extensions, as described above, may be added or inserted to the C- or, N-side of the C- or N-terminal amino acid of the albumin, such as to the C-side of L585 in SEQ ID No. 1.
- The polypeptide may or may not further comprise a further linker to which a conjugation partner, such as a bioactive compound, may be linked. For example a linker may comprise a primary amine such as a lysine.
- One or more conjugation competent cysteines may or may not be created in an albumin by insertion; for example by adding one or more additional cysteines without removal of an amino acid residue from the albumin sequence, or by substituting one or more adjacent amino acids with a larger number of residues containing at least one cysteine, thus extending the overall length of the polypeptide. For example, a cysteine residue may be introduced immediately adjacent an albumin residue identified herein. The cysteine residue may be introduced as a single cysteine residue or within a polypeptide. The polypeptide may be from 2 to 50 amino acids long, preferably from 2, 10, 20, 30, or 40 to 10, 20, 30, 40 or 50 amino acids long.
- Alternatively, or in addition, the invention includes substitution of one of the cysteine residues in one or more disulphides bond of an albumin with a different amino acid residue, so breaking the disulphide bond to leave an additional free thiol group. For example, a cysteine of one or more of the 17 naturally occurring disulphide bonds of HSA may be substituted to provide a conjugation-competent cysteine. Such a substitution causes the cysteine which has not been substituted to no longer have a disulphide binding partner and therefore provide a free thiol group. Conjugation competent cysteines may be provided from one or more of the naturally occurring disulphide bonds of an albumin such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the naturally occurring disulphide bonds of an albumin such as HSA (e.g. SEQ ID No. 1). For example, one cysteine residue which naturally forms a disulphide bond with another cysteine residue may or may not be substituted with a relatively conserved amino acid residue, particularly Ser, Thr, Val or Ala. With reference to SEQ ID No. 1, cysteine residues involved in disulphide bonding are C53, C62, C75, C91, C90, C101, C124, C169, C168, C177, C200, C246, C245, C253, C265, C279, C278, C289, C316, 0361, C360, C369, C392, C438, C437, C448, C461, C477, C476, C487, C514, C559, C558 and C567. Cysteine residues preferred for modification (i.e. deletion or substitution) may in particular correspond to C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567 thus generating a conjugation competent cysteine at one or more of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 of SEQ ID No. 1.
- In addition, conjugation competent cysteines may or may not be created in albumin by deletion of one of the cysteines of a disulphide bond in the protein structure, so breaking the disulphide bond to provide an additional free thiol group.
- Alternatively, or in addition, one or more of the cysteine residues present in the albumin molecule, but not involved in disulphide bonding (e.g. Cys-34 in the case of SEQ ID No. 1) may or may not be deleted (i.e. without substitution with a different amino acid) or may or may not be substituted with a different amino acid, particularly Ser, Thr, Val or Ala.
- For a polypeptide comprising two or more conjugation competent cysteine residues, when the polypeptide is folded, the conjugation competent cysteine residues may or may not be relatively evenly distributed over the surface of the folded protein. The term ‘folded’ includes folding of a polypeptide/protein into its natural configuration, for example the most thermodynamically stable folded configuration. An advantage of relatively even distribution is that it allows conjugation of two or more moieties to the thio-albumin without steric hindrance between two or more of the conjugated moieties. This has the advantage of minimising, and optionally eliminating, potential loss of activity due to issues such as steric hindrance between adjacent moieties (conjugation partners) which may be conjugated to the thio-albumin. Such moieties, for example bioactive molecules, may be relatively bulky.
- Preferably the two or more conjugation competent cysteines are distributed over the surface of the thio-albumin molecule such that they are spaced as far from each other as possible, for example geometrically possible. Preferably the distance between two or more conjugation competent cysteines is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms. Preferably each conjugation competent cysteine is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms distant from all other conjugation competent cysteines in the molecule. The distance between two conjugation competent cysteines is preferably a distance which is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule, for example as shown in a model of an albumin. For example, the longest axis of SEQ ID No. 1 as shown in protein structure 1AO6 is approximately 85 Angstroms. Therefore, it is preferred that the two or more of the cysteine residues are at least 65, 70, 75 or most preferably 80 Angstroms apart. Most preferably each conjugation competent cysteine residue is at a distance of at least 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule.
- Preferably the side chains of conjugation competent cysteines are directed away from each other and/or directed so that a moiety conjugated to the cysteine will be directed away from the centre of the albumin structure. This provides the advantage of preventing interactions between the conjugated moieties and/or the albumin moiety itself.
- With reference to an amino acid sequence, candidate amino acid residues may be visually inspected using software such as PyMOL (Warren L. DeLano “The PyMOL Molecular Graphics System” DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org). Candidate amino acids may be divided into categories based on their proximity to other members of that group. For example, candidate amino acids may be divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 categories. It is preferred that combinations of candidate amino acids are selected from different categories. That is, it is preferred that a thio-albumin contains one or fewer mutations from each category.
- With reference to SEQ ID No. 1, PyMOL was used to analyse the candidate residues of
FIGS. 5A and 5B in order to identify particularly favoured combinations of cysteine mutations. Such combinations may be used to design a thio-albumin having two or more conjugation competent cysteine residues.Selection Groups FIGS. 5A and 5B of the selection method described herein.Selection Group 3 corresponds to the residues identified inFIG. 6B . Particularly favoured residues are given inFIG. 6A and 6B in which the column headings are the same as those inFIG. 1 with the addition of ‘Selection Group’ and ‘Proximity Group’ as described herein. - The results of the analysis are given in
FIG. 10 in which column headings are the same as those used inFIG. 1 , andFIG. 6 with the addition of: - Proximity Group: allocation of a proximity group as described herein to describe subsets of sites within HSA (specifically SEQ ID No. 1).
- For example, candidate amino acid residues selected in Selection Group 1 (listed in
FIG. 5A ) were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members ofSelection Group 1. Five groups were generated (labeled A to E inFIG. 10 ‘proximity group’, right hand column), four were generated by visual inspection. Group E contains amino acid residues not visible in 1AO6 which are known to be in the N-terminal region. In addition it was observed that cys-34 is present in the proximity group A. - Similarly amino acid residues selected in Selection Group 2 (listed in
FIG. 5B ) were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members ofSelection Group 2. Five groups were generated (labeled F-J inFIG. 10 ‘proximity group’, right hand column) - Similarly, the preferred free cysteine residues selected in Selection Group 3 (listed in
FIG. 6B ), which can be generated by mutations causing disruption of disulphide bonds were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of that group. Four groups were generated (labeled K-N inFIG. 10 ‘proximity group’, right hand column). When referring to the residues ofselection group 3, the cited residues are the resultant conjugation competent cysteines (e.g.FIG. 6B ). In order to generate such a conjugation competent cysteine it is clear that the counterpart cysteine (e.g.FIG. 6A ) in the disulphide bond should be removed for example by deletion or substitution. - When a combination of two or more mutations is desired, amino acid residues which occur in different ‘proximity’ groups (e.g. with reference to SEQ ID No.1) may be preferred over those that occur within the same proximity group. For SEQ ID No. 1, there are 14 proximity groups (i.e. A to N). It is preferred that, for a thio-albumin having two or more conjugation competent cysteines, there is zero or one conjugation competent cysteine defined from each of the 14 groups. That is, it is preferred that such a thio-albumin does not contain two or more conjugation competent cysteines falling within the same group. A large number of permutations exist which meet this criterion.
- For example, for a thio-albumin variant containing two free thiol groups residues based on selection criteria that generated
Selection Group 1, then T79+A364, in which one residue is selected from proximity group A to combine with A364 in proximity group C, would be preferred over T79+E86 which both occur in proximity group A. - For combinations including cysteine-34, it is preferred not to select residues from proximity groups A, F or K. That is, it is preferred to select residues from one or more of proximity groups B to E, G to J and L to N.
- Examples of preferred mutations selected from within
Selection Group 1 may include the following: -
- For 2 amino acid residues selected from
Selection Group 1; amino acid residues from proximity groups A+C are preferred, such as T79+A364, Similarly, amino acid residues selected from proximity groups C+E, such as A364+D1 are also preferred. Also, amino acid residues from proximity groups D+E, such as L585+A2 or + the C-side of L585+A2, or from G+I, such as S270+A581, are preferred. - For 3 amino acid residues selected from
Selection Group 1; amino acid residues which occur in proximity groups A+C+B are preferred such as T79+A364+D562. Similarly, amino acid residues selected from proximity groups B+C+E, such as D562+A364+A2 or D562+A364+D1, are also preferred - For 4 amino acid residues selected from
Selection Group 1; amino acid residues which occur in proximity groups A+C+B+D such as such as T79+A364+D562+A504, or alternatively T79+D562+A364+L585 are preferred. Even more preferred are amino acid residues selected from proximity groups A+B+C+E, such as C34+D562+A364+A2 T79+D562+A364+D1. - For 5 amino acid residues selected from
Selection Group 1; amino acid residues which occur in proximity groups A+C+B+D+E such as T79+D562+A364+L585+D1 are preferred. Similarly, E86+D562+A364+A504+A2 are also preferred. - The above mentioned albumin variants may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position in another albumin.
- For 2 amino acid residues selected from
- Examples of preferred mutations selected from within
Selection Group 2 may include the following: -
- For 2 amino acid residues selected from
Selection Group 2; amino acid residues which occur in proximity groups G+I such as S270+A581 are preferred. Alternatively, amino acid residues which occur in proximity groups G+H such as S270+D129 are preferred. - For 3 amino acid residues selected from
Selection Group 2; amino acid residues which occur in proximity groups G+I+F such as S270+A581+E82 are preferred. Alternatively, amino acid residues which occur in proximity groups G+I+H such as S270+A581+D129 are preferred. - For 4 amino acid residues selected from
Selection Group 2; amino acid residues which occur in proximity groups G+I+F+H such as S270+A581+E82+D129. - For 5 amino acid residues selected from
Selection Group 2; amino acid residues which occur in proximity groups G+I+F+H+J such as S270+A581+E82+D129+Q397 are preferred. However, D549 is not preferred in combination with mutations A578, A581. Also, mutations to D549, A578, A581 or are not preferred in combination with mutation of L585 fromSelection Group 1.
- For 2 amino acid residues selected from
- Examples of preferred site selected from within
Selection Group 3 for the conjugation competent free-thiols may include the following: -
- For 2 amino acid residues selected from
Selection Group 3; amino acid residues which occur in proximity groups M+L are preferred, such as C369+C177. Similarly, C361+C124 are also preferred. - More than two mutations disrupting disulphide bonds are less preferred.
- The above mentioned albumin variants may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position in another albumin.
- For 2 amino acid residues selected from
- Combinations of sites from
Selection Groups Selection Group 1 are typically preferred to sites fromSelection Group 2, which are typically preferred to sites selected fromSelection Group 3. - Examples of sites from
Selection Groups 1+2 may include residues from proximity groups C+I, such as A364+A581. Alternatively, residues from proximity groups A+G+I, such as C34+S270+A581, from proximity groups A+H+G+I, such as C34+D129°S270+A581, from proximity groups A+C+I, such as T79+A364+A581, or residues from proximity groups C+I+H such as A364+A581+D129 are also preferred. - Examples of sites from
Selection Groups 1+3 may include residues from proximity groups A+L+M, such as C34+C169+C316, from proximity groups C+L, such as A364+C177 are preferred. Alternatively, residues from proximity groups B+M, such as D562+C369 are preferred. - Examples of sites from
Selection Groups 2+3 may include residues from proximity groups H+M, such as D129+C369 are preferred. Alternatively, residues from proximity groups I+M, such as A581+C369 are preferred. - Examples of sites from
Selection Groups 1+2+3 may include residues from proximity groups A+H+M+D, such as C34+D129+C360+L585, from proximity groups B+H+M, such as D562+D129+C369 are preferred. - The above combinations are generally more preferred than combinations of residues from the following sets of proximity groups: (i) A, K and F; (ii) B, D, I, J and N; (iii); C and M; (iv) H and L.
- The above albumin variants of the invention may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position, if based on an albumin other than SEQ ID No. 1.
- A skilled person will appreciate that the sites for introduction of more than one free thiol group (conjugation competent cysteine) have been selected from
Selection Groups - A preferred thio-albumin comprises SEQ ID No. 1 with Cys at
positions positions positions - The polypeptide may or may not comprise at least one mutation that reduces glycosylation.
- A third aspect of the invention provides a polynucleotide which encodes the polypeptide according to the invention. The polynucleotide may or may not be codon-optimised relative to the host from which it is to be expressed. SEQ ID No. 2 provides the usual coding sequence of HSA (SEQ ID No. 1). SEQ ID No. 3 provides a coding sequence of HSA (SEQ ID No. 1) which is codon-optimised for expression from S. cerevisiae. SEQ ID No. 2 or 3 may be mutated in order to provide a polynucleotide which encodes a polypeptide according to the invention. Preferably the polynucleotide is synthetic and/or recombinant. Preferably the polynucleotide is an isolated polynucleotide. The polynucleotide may encode an HSA with or without a leader sequence. For example, the polynucleotide may encode an HSA with the natural leader sequence of HSA (
amino acids 1 to 24 of SEQ ID No. 102) or an HSA with a fusion leader sequence (amino acids 1 to 24 of SEQ ID No. 49). - A fourth aspect of the invention provides a plasmid comprising the polynucleotide of the third aspect of the invention. The plasmid may be a 2 micron based plasmid such as those described in WO2005/061719, WO2005/061718 and WO2006/067511 (all incorporated herein by reference). The plasmid may exhibit enhanced chaperone activity, for example through over expression of a chaperone, particularly PDI.
- A fifth aspect of the invention provides an expression system such as a host cell comprising a polynucleotide according to the third aspect of the invention and/or a plasmid of the fourth aspect of the invention. Preferably the host cell is a mammalian cell such as a human or bovine cell, or a fungal cell such as a yeast cell. Alternatively, the host cell may be a bacterial cell such as a Bacillus or Escherichia coli or a viral cell such as Baculovirus or a plant cell such as a rice e.g. Oryza sativa. Most preferably, the cell is a yeast cell such as a Saccharomyces (e.g. S. cerevisiae), a Pichia or an Aspergillus cell.
- A sixth aspect of the invention provides a conjugate which comprises a conjugation partner, such as a bioactive compound, and a polypeptide according to the invention, wherein the conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide. The conjugation partner may be a therapeutic, diagnostic or imaging compound such as those mentioned herein. The conjugate may comprise 2 or more, for example 2, 3, 4, 5, 6, 7,8, 9 or 10, conjugation partners which may each be different and/or may be multiple copies of the same compound. Preferably, each conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide, however conjugation partners may be linked by other means for example by a genetic fusion or covalent bonds to non-cysteine amino acids such as lysine.
- A seventh aspect of the invention provides a method of producing a polypeptide of the invention comprising:
- (a) culturing a host cell according to the invention under conditions that allow expression of the polypeptide; and
- (b) recovering the polypeptide from the host cell and/or from host cell growth medium.
- Accordingly, the present invention also provides a method for producing a polypeptide (or protein) of the invention, the method comprising: (a) providing a host cell of the invention comprising a polynucleotide encoding protein product of choice as defined above; and (b) growing the host cell (for example, culturing the host cell in a culture medium); thereby to produce a cell culture or recombinant organism comprising an increased level of the protein product of choice compared to the level of production of the protein product of choice achieved by growing (for example, culturing), under the same conditions, the same host cell that has not been genetically modified to cause over-expression of one or more helper proteins.
- The step of growing the host cell may or may not involve allowing a host cell derived from a multicellular organism to be regrown into a multicellular recombinant organism (such as a plant or animal) and, optionally, producing one or more generations of progeny therefrom.
- The method may or may not further comprise the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.
- The production method may comprise linking a conjugation partner to the polypeptide of the invention through a conjugation competent cysteine residue of the polypeptide. Suitable conjugation methods and conjugation partners are described herein.
- An eighth aspect of the invention provides a composition comprising a conjugate according to the invention and at least one pharmaceutically acceptable carrier and/or diluent.
- A ninth aspect of the invention provides a method for making a pharmaceutical ingredient and/or a pharmaceutical product comprising making a thio-albumin according to the present invention, optionally conjugating a further molecule to the thio-albumin, optionally formulating the resultant conjugate with a pharmaceutically acceptable diluent and/or carrier and optionally preparing the product in unit dosage form.
- A tenth aspect of the invention provides use of a polypeptide according to the invention for the production of a thio-albumin-conjugate.
- An eleventh aspect of the invention provides use of a conjugate according to the invention and/or produced by a method according to the invention for treatment of disease, treatment of illness and/or diagnosis.
- A twelfth aspect of the invention provides a gel comprising one or more albumins according to the invention. The gel may be formed by any suitable method. For example the gel may be formed by incubating an albumin solution, or suspension, at a suitable temperature e.g. room temperature (15 to 25° C., such as 20° C.) or body temperature (36 to 38° C., preferably 36.9° C.). A gel may be used to coat medical devices, such as a stent. A gel may be used in or on a wound dressing. The albumin may be applied to the medical device or wound dressing before or after it has gelled. The albumin may be applied ex situ or in situ (e.g. applied to a medical device or dressing before, after or during its application on to or insertion into a human or animal body).
- The polypeptides and/or conjugates of the invention may be used to prepare nanoparticles which may be used, for example, in angiogenic applications, anti-angiogenic applications and to coat a medical device such as a stent. Nanoparticles are effective at targeting, for example to non tight-junctions, and therefore can be useful for targeting tumours such as cancerous tumours. Nanoparticles can also be useful to target antigen in order to provoke an immune response since nanoparticles are particularly susceptible to engulfment and presentation by phagocytes. The invention provides nanoparticles consisting only of thio-albumin according to the invention which may or may not be conjugated to a moiety (conjugation partner). The invention also provides nanoparticles comprising thio-albumin according to the invention, which may or may not be conjugated to a moiety, and one or more other constituents of a nanoparticle which may or may not be albumin related. In a preferred embodiment, a thio-albumin according to the invention comprises at least two conjugation competent cysteine residues located on the surface of the polypeptide. Such a thio-albumin may be used for the preparation of nanoparticles in which one or more conjugation competent cysteine residues may be used in the formation of a nanoparticle and one or more conjugation competent residue is used for conjugation to a conjugation partner, for example to a bioactive molecule.
- The invention relates to all albumins. Whilst preferred residues have been identified in relation to SEQ ID No. 1, the skilled person would be able to identify equivalent residues in other albumin sequences, such as the albumins disclosed in
FIGS. 2 and 3 , and understand that mutations of albumins (other than SEQ ID No. 1) at such equivalent residues are part of the invention. Equivalent residues can be identified by, for example, homology alignment with SEQ ID No. 1. A residue in an albumin other than SEQ ID No. 1 may or may not have an identical residue coordinate to its equivalent residue in SEQ ID No. 1. Thus the invention provides thio-albumins based on any albumin sequence, such as the sequences shown in Table 1 and, more preferably, those shown inFIG. 2 and/or 3 . ‘Based on’ includes modification of an albumin sequence to introduce one or more additional free-thiols. - Recombinant albumins can offer advantages over animal-derived albumins by having a higher level of conjugation-competent free thiol groups,' and can be manufactured without the risk of contamination with pathogenic prions and viruses. An advantage of a thio-albumin conjugate is that the thio-albumin part may be prepared separately to a conjugation partner. Therefore, one batch of thio-albumin may be used to produce many different thio-albumin conjugates. Also, the individual components of the conjugate can be manufactured by different methods and therefore are not restricted to a single method, such as heterologous protein expression in a host cell such as a yeast. Furthermore, a thio-albumin may comprise multiple conjugation sites and therefore a single thio-albumin may be conjugated to more than one type of conjugation partner (e.g. therapeutic agent, diagnostic agent, targeting agent, imaging agent) and/or to multiple copies of one or more types of conjugation partner. The ability to conjugate the thio-albumin to different types of conjugation partners allows the provision of a multi-functional species. The ability to conjugate the thio-albumin to multiple copies of a conjugation partner allows the concentration of molecule to be increased and therefore increase the amount, or volume, of thio-albumin conjugate required for a given purpose relative to a conjugate having only a single copy of the conjugation partner. Advantages of delivering drugs via an albumin fusion protein are discussed in Osborn, et al. (2002). J Pharmacol Exp Ther 303(2): 540-8. It is expected that delivery of drugs via a conjugation of the invention would have similar advantages.
- Further details which may or may not be used in accordance with the invention are described below:
- The above disclosure has been made in relation to the albumin model known as 1AO6 (Protein Data Bank) which relates to SEQ ID No. 1.
FIG. 1 gives the amino acid residues for 1AO6. - However, the invention relates to all albumins and their structures. Structures of albumin are available to the skilled person, for example the atomic coordinates for the tertiary structure of human albumin are available at the GenBank DNA database at www.ncbi.nlm.nih.gov.
- Structures may be viewed using suitable software such as RasM.1 Chime (Sayle,
TIBS -
- 1AO6, 1BMO (Sugio et al. (1999). Protein Enq 12(6): 439-46), which was among the top 17 requested proteins.
- 1 UOR, He & Carter (1992). Nature 358(6383): 209-15.
- 1bj5 and 1bke, Curry et al. (1998). Nat Struct Biol 5(9): 827-35.
- 1e7a,1e7b, 1e7c, Bhattacharya et al. (2000). J Biol Chem 275(49): 38731-8.
- 1e7e, 1e7f, 1e7g, 1e7h and 1e7i, Bhattacharya et al. (2000). J Mol Biol 303(5): 721-32.
- 1GNJ, Petitpas et al. (2001). J Mol Biol 314(5): 955-60.
- 1HA2 and 1H9Z Petitpas et al. (2001). J Biol Chem 276(25): 22804-9.
- The albumin used in the invention may be a naturally occurring albumin, an albumin-related protein or a variant thereof such as a natural or engineered variant. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins. An albumin, of this invention, may comprise the sequence of an albumin protein obtained from any source. Typically the source is mammalian such as human or bovine. In one preferred embodiment the serum albumin is human serum albumin (“HSA”). The term “human serum albumin” includes a serum albumin having an amino acid sequence naturally occurring in humans, and variants thereof. Preferably the albumin has the amino acid sequence of SEQ ID No. 1 or a variant or fragment thereof, preferably a functional variant or fragment thereof. The HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example,
EP 0 073 646 andEP 0 286 424. A fragment or variant may or may not be functional. For example, a fragment or variant may retain the ability to bind to an albumin receptor such as FcRn to at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the ability of the parent albumin (from which the fragment or variant derives) to bind to the receptor. Relative binding ability may be determined by methods known in the art such as surface plasmon resonance studies. - The albumin may be a naturally-occurring polymorphic variant of human albumin or of a human albumin analogue. Generally, variants or fragments of human albumin will have at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, (preferably at least 80%, 90%, 95%, 100%, 105% or more) of human albumin's ligand binding activity (for example FcRN-binding), mole for mole.
- The “albumin” may comprise the sequence of bovine serum albumin. The term “bovine serum albumin” includes a serum albumin having an amino acid sequence naturally occurring in cows, for example as taken from Swissprot accession number P02769, and variants thereof as defined herein. The term “bovine serum álbumin” also includes fragments of full-length bovine serum albumin or variants thereof, as defined herein.
- A number of proteins are known to exist within the albumin family; a non-exclusive list is shown in Table 1, below. The list indicates full-length of sequences including the mature protein and leader sequence (unless indicated otherwise).
- The albumin may comprise the sequence of an albumin derived from one of serum albumin from dog (e.g. see Swissprot accession number P49822-1), pig (e.g. see Swissprot accession number P08835-1), goat (e.g. as available from Sigma as product no. A2514 or A4164),), cat (e.g. see Swissprot accession number P49064-1), chicken (e.g. see Swissprot accession number P19121-1), ovalbumin (e.g. chicken ovalbumin) (e.g. see Swissprot accession number P01012-1), turkey ovalbumin (e.g. see Swissprot accession number O73860-1), donkey (e.g. see Swissprot accession number Q5XLE4-1), guinea pig(e.g. see Swissprot accession number Q6WDN9-1), hamster (see DeMarco et al. (2007). International Journal for Parasitology 37(11): 1201-1208), horse (e.g. see Swissprot accession number P35747-1), rhesus monkey (e.g. see Swissprot accession number Q28522-1), mouse (e.g. see Swissprot accession number P07724-1), pigeon (e.g. as defined by Khan et al, 2002, Int. J. Biol. Macromol., 30(3-4),171-8), rabbit (e.g. see Swissprot accession number P49065-1), rat (e.g. see Swissprot accession number P02770-1) and sheep (e.g. see Swissprot accession number P14639-1) and includes variants and fragments thereof as defined herein.
- The albumin may comprise the sequence of an albumin such as a serum albumin or an ovalbumin, for example those shown in Table 1, below, and includes variants and fragments thereof as defined herein.
-
TABLE 1 Albumins from various species Identity to SwissProt SEQ ID Accession NO: 1 Length Protein Common Name Species No (Clustal V) (aa) SA African clawed Xenopus laevis P08759-1 37.3 608 frog SA Bovine Bos taurus (SEQ P02769-1 76.1 607 ID No. 94) SA Cat Felis catus (SEQ P49064-1 82.2 608 ID No. 95) SA Chicken Gallus gallus P19121-1 46.5 615 (Version 2 text) SA Cobra ALB Naja kaouthia Q91134-1 30.9 614 SA Dog Canis lupus P49822-1 80.3 608 familiaris (SEQ ID No. 96) SA Donkey Equus asinus Q5XLE4-1 76.6 607 (SEQ ID No. 97) SA European water Rana shqiperica Q9YGH6-1 31.5 603 frog SA Blood fluke Schistosoma AAL08579 76.0 608 mansoni Q95VB7-1 SA Mongolian Gerbil Meriones O35090-1/ 73.2 609 unguiculatus (SEQ JC5838 ID No. 98) SA Goat Capra hircus (SEQ B3VHM9-1 74.3 607 ID No. 99) SA Guinea Pig Cavia porcellus Q6WDN9-1 73.0 608 (SEQ ID No. 100) SA Horse Equus caballus P35747-1 76.4 607 (SEQ ID No. 101) SA Human Homo sapiens P02768-1 100.0 609 (SEQ ID No. 102) SA Australian Neoceratodus P83517-1 22.8 101 Lungfish fosteri (NL) SA Macaque Macaca mulatta Q28522-1 93.5 608 (Rhesus (SEQ ID No. 103) Monkey) SA Mouse Mus musculus P07724-1 72.4 608 (SEQ ID No. 104) Version 3. SA North American Rana catesbeiana P21847-1 36.1 382 bullfrogs (NL) SA Pig Sus scrofa (SEQ P08835-1 75.6 607 ID No. 105) Version 2SA Rabbit Oryctolagus P49065-1 75.3 608 cuniculus ( SEQ ID Version 2 No. 106) SA Rat Rattus norvegicus P02770-1 73.4 608 (SEQ ID No. 107) Version 2.SA Salamander Ambystoma Q8UW05-1 38.8 626 maculatum SA Salmon ALB1 Salmo salar P21848-1 25.0 608 SA-2 Salmon ALB2 Salmo salar Q03156-1 24.8 608 SA Sea lamprey Petromyzon Q91274-1 16.9 1423 marinus SA Sea lamprey Petromyzon O42279-1 17.4 551 marinus -AS SA Sheep Ovis aries (SEQ ID P14639-1 75.3 607 No. 108) SA Sumatran Pongo abelii Q5NVH5-1 100.0 609 Orangutan SA Tuatara Sphenodon Q8JIA9-1 43.8 527 punctatus (NL) SA Western clawed Xenopus (Silurana) Q6DJ95-1 10.5 572 frog tropicalis (NL) OA Chicken Gallus gallus P01012-1 10.9 383 Version 2 (NL) OA Turkey Meleagris O73860-1 11.4 386 gallopavo Version 3. (NL) SA: Serum albumin, SA-2: Serum albumin-2, OA: Ovalbumin, NL: No Leader sequence - Many naturally occurring mutant forms of albumin are known. Many are described in Peters, (1996, All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, Inc., San Diego, Calif., p.170-181). A variant as defined herein may be one of these naturally occurring mutants such as those described in Minchiotti at al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768,.
- A “variant albumin” refers to an albumin protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin or a fatty acid such as a long-chain fatty acids, for exampleoleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and/or palmitoleic (C16:1)), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein, e.g. the protein from which the variant is derived. Such characteristics may be used as additional selection criteria in the invention.
- Typically an albumin variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with a naturally occurring albumin such as SEQ ID No. 1. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program or the Clustal V program and therefore allow calculation of % homology between sequences of a multiple alignment and/or calculation of % identity between sequences of a pairwise alignment. The parameters used may be as follows:
- Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM Custal W: Pairwise alignment parameters: ‘Slow-Accurate’, Gap Penalty: 10, Gap Length: 0.1, Protein Weight Matrix:
Gonnet 250, DNA Weight Matrix: IUB. Multiple Alignment Parameters: Gap penalty 10.00, gap length penalty 0.20, Delay Divergent Seqs(%) 30, DNA transition weight 0.50, Protein weight matrix=Gonnet series, DNA weight matrix=IUB. - Clustal V: Pairwise alignment parameters: Ktuple: 1, Gap Penalty: 3, Window: 5, Diaganols: 5; Multiple alignment parameters:
Gap penalty 10,gap length penalty 10. - As used herein, the term “conservative” amino acid substitutions refers to substitutions made within the same group, and which typically do not substantially affect protein function. By “conservative substitutions” is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made by techniques well known in the art, such as by site-directed mutagenesis as disclosed in U.S.4 Pat. No 4,302,386 issued 24 Nov. 1981 to Stevens, incorporated herein by reference.
- In one embodiment, the Venn diagram of
FIG. 4 may be used to determine conservative amino acid substitutions: UsingFIG. 4 ., a conservation mutation score (ranging from 0 to 5) may be calculated. A score of 0 is the highest conservation, which, for cysteine, is only assigned for substitution of a cysteine residue with another cysteine residue. For changes from any other amino acid to a cysteine, the score may be 1, 2, 3, 4, 5. A score of 1 is a more conservative substitution that a score of 2, 3, 4 or 5. A score of 5 is assigned to the lowest conservation between a substituted amino acid and the cysteine. The score of 0 to 5 is calculated fromFIG. 4 as the number of boundaries (i.e. lines) crossed to go from cysteine to the appropriate amino acid. Thus the score for cysteine is 0 as no boundaries are crossed. Likewise, the score of aspartic acid (D) is 3, since 3 boundaries are crossed. - The conservation mutation score (with respect to
FIG. 4 ) for the 20 different amino acids are defined as (using one-letter codes for the amino acids): A=1, C=0, D=3, E=4, F=4, G=2, H=5, I=4, K=4, L=4, M=3, N=2, P=3, Q=3, R=5, S=1, T=1, V=3, W=3, Y=3. With reference toFIGS. 1, 5A, 5B, 5C, and 5D , these scores are provided for each of the amino acid residues in the column labelled ‘Conserved Mutation to Cysteine’. Using the conservation mutation score residues with a score of 3 or less, i.e. aspartic acid methionine, proline, glutamine, valine, tryptophan, tyrosine, glycine, asparagine, alanine, serine and threonine are preferred since they are relatively conserved with cysteine. More preferred are those amino acids with a score of 2 or less i.e. glycine, asparagine, alanine, serine, threonine. Most preferred are those with a score of 1, i.e. alanine, serine, threonine. Similarly using the conservation mutation score system ofFIG. 4 , residues with a score of 4 or more, i.e. glutamic acid, phenylalanine, isoleucine, lysine, leucine, histidine and arginine are less preferred and may not be preferred at all. - Alternatively, or in addition, “conservative” amino acid substitutions refers to substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
- For example, a conservative substitution of alanine-2 in
SEQ ID No 1 can include glycine or serine. Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group. For example, a non-conservative substitution could include the substitution of a polar amino acid for a hydrophobic amino acid. - The term “fragment” as used herein includes any fragment of full-length albumin or a variant thereof, so long as at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein. A fragment will typically be at least 50 amino acids long. A fragment may comprise at least one whole sub-domain of albumin. Domains of HSA have been expressed as recombinant proteins (Dockal et al., 1999, J. Biol. Chem., 274, 29303-29310), where domain I was defined as consisting of amino acids 1-197, domain II was defined as consisting of amino acids 189-385 and domain III was defined as consisting of amino acids 381-585. Partial overlap of the domains occurs because of the extended a-helix structure (h10-h1) which exists between domains I and II, and between domains II and III (Peters, 1996, op. cit., Table 2-4). HSA also comprises six sub-domains (sub-domains IA, IB, IIA, IIB, IIIA and IIIB). Sub-domain IA comprises amino acids 6-105, sub-domain IB comprises amino acids 120-177, sub-domain IIA comprises amino acids 200-291, sub-domain IIB comprises amino acids 316-369, sub-domain IIIA comprises amino acids 392-491 and sub-domain IIIB comprises amino acids 512-583. A fragment may comprise a whole or part of one or more domains or sub-domains as defined above, or any combination of those domains and/or sub-domains. A fragment may comprise or consist of at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of an albumin or of a domain of an albumin.
- Additionally, single or multiple heterologous fusions comprising any of the above; or single or multiple heterologous fusions to albumin, or a variant or fragment of any of these may be used. Such fusions include albumin N-terminal fusions, albumin C-terminal fusions and co-N-terminal and C-terminal albumin fusions as exemplified by WO 01/79271.
-
FIGS. 2 and 3 show alignments of various albumin family proteins with HSA (SEQ ID NO: 1), identified as ‘P02768’. The protein sequences include the albumin leader sequence. These alignments can be used to identify conserved regions and amino acid residues corresponding to those in HSA selected as described above. One or both alignments can also be used to assign a homology score to an amino acid residue in an albumin sequence. - An example of such a procedure is the MegAlign program (version 8.0.2) developed by DNASTAR Inc., part of the Lasergene suite, based on Hein, J. J. (1990) “Unified approach to alignment and phylogenies.” In Methods in Enzymology, Vol. 183: pp. 626-645. Using the Jotun Hein Method and the settings GAP PENALTY=11, GAP LENGTH PENALTY=3 for multiple alignments and KTUPLE=2 for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal V Method and the settings GAP PENALTY=10,
GAP LENGTH 10 for multiple alignments and KTUPLE=1. GAP PENALTY=3, WINDOW=5 and DIAGONAL=5 for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal W Method and the settings GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DIVERGENCE=30 DNA transition=0.5 and using GONNET SERIES for Protein Weight matrix and IUB for DNA Weight matrix for multiple alignments, and Slow accurate, GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and using GONNET SERIES for Protein Weight matrix and IUB for DNA Weight matrix for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal V method may be used (above).The alignment of two amino acid sequences may also be determined by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman and Wunsch (1970) “A general method applicable to the search for similarities in the amino acid sequence of two proteins.” J. Mol. Biol. 48, 443-453. The substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5. -
FIG. 2 shows an alignment of sixteen mammalian albumin family proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal W. The protein sequences include the albumin leader sequence. Each sequence is labelled with the animal from which it derives and its database accession number. -
FIG. 3 shows alignments of thirty three albumin family (both mammalian and non-mammalian) proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal V. The protein sequences include the albumin leader sequence. - Homology may be determined with reference to
FIG. 2 and/orFIG. 3 . The degree of identity between a given amino acid sequence and SEQ ID NO: 1 may be calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the shorter of the two sequences. The result may be expressed in percent identity. An exact match occurs when the two sequences have identical amino acid residues in the same positions of the overlap. The length of a sequence is the number of amino acid residues in the sequence. - In addition, individual amino acid residues of HSA can be ranked according to their conservation to the amino acids of other albumin family proteins at the same position.
FIG. 1 , column labelled ‘Align 1 (Mamm. W ('mammalian, Clustal W)) provides the homology level for each position of SEQ ID No. 1 as calculated by the alignment of mammalian albumins given inFIG. 2 . The homology level score may be calculated. One method to score the homology level, which is used herein, is calculated by using the strength of the histogram provided by MegAlign program (version 8.0.2) (ranging 0 to 100%); six levels of homology are determined with the highest=100%, decreasing in 20% increments to the lowest (0%) and shown by bar height inFIG. 2 . A score of 100 is the highest conservation and indicates there are no changes at that residue when the sequence from human serum albumin is compared with other mammalian albumin sequence, whereas a score of 0 indicates the lowest level of conservation between the aligned sequences. - Similarly, the homology level score for each amino acid residue in HSA (
FIG. 1 , column labelled ‘Align 2 (Var. Sps. (‘various species (i.e. mammalian and non-mammalian), Clustal V’)) calculated using the strength of the histogram provided by Megalign when various albumins (including non-mammalian albumins) are aligned using Clustal V. A person skilled in the art will appreciate that a range of different albumin sequences and alignment algorithms may be used to calculate the homology level score. - When using homology scores, such as those described in reference to the alignments of
FIGS. 2 and 3 , to identify candidate residues for the present invention, preferred residues include those which are not highly conserved (for example those with a score of less than 40, more preferably less than 20 and most preferably 0) are preferred and those with a higher level of homology (for example those with a score of more than 40, more than 60, more than 80 and most preferably 100) are less preferred. - Each of the amino acid residue in HSA (SEQ ID No. 1) were scored according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with mammalian albumins (
FIG. 1 , column labelled ‘Adj. 100%'s (Align 1). This is because if an amino acid is within a highly conserved domain, it may be important to the structure of function of the protein and thus disruption may be undesirable. InFIG. 1 , a score of 0 indicates the residue is not adjacent to any residue with 100% homology (with reference to the alignment ofFIG. 2 ) when HSA is aligned with mammalian albumins; a score of 1 indicates that the residue is adjacent to one residue with 100% homology when HSA is aligned with mammalian albumins; a score of 2 indicates that the residue is adjacent to two residues with 100% homology when HSA is aligned with mammalian albumins. Residues with a score of 0 or 1 are preferred. Residues with a score of 0 are most preferred. - For example, amino acid residues with a score of 2 (such as valine-7 (V7)) are preferably deselected using the method of the invention since these amino acid residues were assumed to occur in a region of high homology which would be unlikely to accept a mutation to an alternative amino acid. Similarly, phenylalanine-11 (F11) is adjacent to one 100% conserved residue, in a region of conserved residues, and is less preferred to a residue, such as alanine-2 (A2), which has no adjacent 100% conserved residues.
- In accordance with the invention, additional information such as preferred sites for insertion, deletions or substitutions may also be obtained by alignment analysis. For example, mouse albumin contains 36 cysteine residues, all the cysteines involved in disulphide bonding (by homology to HSA) are present, as is cysteine-34, however a cysteine residue is present at 579 on mature mouse protein but not other mammalian albumin sequences therefore thio-albumin mutein S579C may be preferred as its lack of homology with other mammalian albumins suggests that it may not be particularly important to the structure and/or function of this albumin.
- In addition, using the alignments of various mammalian albumin family and Clustal W (
FIG. 2 ) shows that compared to other mammalian albumins, gerbil albumin has an additional alanine residue between alanine-2 (A2) and histidine-3 (H3), indicating that insertion of a cysteine residues after residue 2 (e.g. A2 of SEQ ID No. 1) and before residue 3 (e.g. H3 of SEQ ID No. 1) is preferred. - Compared to other mammalian albumins, guinea pig albumin has a serine residue at cysteine-34 (C34). In examples where deletion of the free thiol group at cysteine-34 is required, a mutation such as C34S may be preferred.
- Most mammalian albumin sequences (with the exception of human serum albumin) have a sequence which is less than or equal to 584 amino acids in length (less than or equal to 608 amino acids including leader sequence). Using the alignment in
FIG. 2 the additional amino acid residue present on human serum albumin appears to be at the C-terminus without any cognate alignment amino acid residues in the other mammalian serum albumin sequences. Thus a thio-albumin variant containing G584C and a deletion of L585 may be preferred. - A number of albumin sequences (Bovine, Donkey, Goat, Horse, Sheep, Pig)are 583 amino acids in length (607 amino acids including leader sequence). Using the alignment in
FIG. 2 , it can be seen that these species albumin sequences do not have a residue corresponding R117 (R141 including leader sequence) therefore a thio-albumin containing V116C and a deletion of R117 or a thio-albumin containing a deletion of R117 and P118C may be preferred. In such a thio-albumin the length of the amino acid sequence would be reduced relative to SEQ ID No. 1. - The alignment used in
FIG. 2 and the conclusions drawn are in particular with reference to mammalian albumins and Clustal W, the skilled person will appreciate that the teaching applies likewise to other members of the albumin family and to alternative alignment algorithms. - The albumin variant may have at least 40% identity with SEQ ID NO: 1, particularly at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity.
- Identity may be calculated using any method, for example those described herein.
- The thio-albumin may optionally be fused to one or more conjugation partners for example through a genetic or chemical fusion. For a thio-albumin which comprises a genetic fusion, the fusion may be at the N- or C-terminus or comprise an insertion.
- With respect to genetic fusions of albumin, the skilled person will also appreciate that the open reading frame of any other gene or variant, or part or either, can be utilised as an open reading frame for use with the present invention. For example, the open reading frame may encode a protein comprising any sequence, be it a natural protein (including a zymogen), or a variant, or a fragment (which may, for example, be a domain) of a natural protein; or a totally synthetic protein; or a single or multiple fusion of different proteins (natural or synthetic). Such proteins can be taken, but not exclusively, from the lists provided in WO 01/79258, WO 01/79271, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, or a variant or fragment thereof; the disclosures of which are incorporated herein by reference. Although these patent applications present the list of proteins in the context of fusion partners for albumin, the present invention is not so limited and, for the purposes of the present invention, any of the proteins listed therein may be presented alone or as fusion partners for albumin or any other protein or fragment or variant of any of the above, as a desired polypeptide. Examples of chemical fusions (also known as conjugations) of albumin are given in Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; and Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8.
- An advantage of using a genetically or chemically fused albumin is that either or all of the molecules which contribute to the fusion may have improved properties relative to the unfused molecule(s) (Balan et al. (2006) Antivir Ther 11(1): 35-45). Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz, F. (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused conjugation partner, for example bioactive molecule, from inactivation.
- The polypeptide may display modified (e.g. reduced) glycosylation, such as, but not limited to reduced N-linked glycosylation or reduced O-linked glycosylation. The N-linked glycosylation pattern of an albumin molecule can be modified by adding/removing amino acid glycosylation consensus sequences such as N-X-S/T, at any or all of the N, X, or S/T position. Albumin polymorphisms exist with N-linked glycosylation. Albumin mutants may have recycling time such that the efficacy of a mutant as a bioactive carrier is improved. Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell. For example, the albumin protein sequence of SEQ ID No. 1 does not contain any sites for N-linked glycosylation and has not been reported to be modified, in nature, by O-linked glycosylation. However, it has been found that recombinant human albumin (“rHA”) produced in a number of yeast species can be modified by O-linked glycosylation, generally involving mannose. The mannosylated albumin is able to bind to the lectin Concanavalin A. The amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced. Alternatively, the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
- If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia pastoris or Kluyveromyces lactis. The sequence of PMT1 (or any other PMT gene) isolated from S. cerevisiae may be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species. The cloning of the PMT1 homologue of Kluyveromyces lactis is described in WO 94/04687.
- The step of “purifying the thus expressed heterologous protein from the cultured host cell or the culture medium” optionally comprises cell immobilization, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilization, separation and/or breakage.
- Cell immobilization techniques, such as encasing the cells using calcium alginate beads, are well known in the art. Similarly, cell separation techniques, such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like are well known in the art. Likewise, methods of cell breakage, including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
- The at least one other purification step may be any other step suitable for protein purification known in the art. For example purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye;
EP 464 590, removal of yeast-derived colorants;EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference. - The thio-albumin or fusions of thio-albumin and another protein or proteins can be prepared by methods know to the art (Sanker, (2004), Genetic Eng. News, 24, 22-28, Schmidt, (2004), Appl. Microbiol. Biotechnol., 65, 363-372) including but not limited to expression in mammalian cell culture (Mason et al., (2004), Protein Expr. Purif., 36, 318-326; Mason at al., (2002), Biochemistry, 41, 9448-9454) from cells lines such as CHO (and its variants), NS0, BHK, HEK293, Vero or PERC6 cells by transformation or transient expression; insect cell culture (Lim et al., (2004) Biotechnol. Prog., 20, 1192-1197); plant cell culture from such plants as Lemna or Oryza sativa; transgenic animals (Dyck et al., (2003)
- Trends in Biotechnology, 21, 394-399); transgenic plants (Ma et al., (2003) Nature Reviews Genetics, 4, 794-805); Gram positive and Gram negative bacteria such as Bacillus and Escherichia coli (Steinlein, and Ikeda, (1993), Enzyme Microb. Technol., 15, 193-199); filamentous fungi including but not restricted to Aspergillus spp (EP 238023, U.S. Pat. No. 5,364,770, U.S. Pat. No. 5,578,463, EP184438, EP284603, WO 2000/056900, WO9614413), Trichoderma spp and Fusarium spp (Navalainen at al., (2005), Trends in Biotechnology, 23, 468-473).
- The host cell may be any type of cell. The host cell may or may not be an animal (such as mammalian, avian, insect, etc.), plant (such as Oryza sativa), fungal or bacterial cell. Bacterial and fungal, such as yeast, host cells may or may not be preferred.
- Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).
- A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, N.J., USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
- Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls. One such method involves ligation via cohesive ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
- A further method uses synthetic double stranded oligonucleotide linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3′ termini and fill in recessed 3′ ends. Synthetic linkers and pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end. Alternatively a DNA fragment or DNA fragments can be ligated together by the action of DNA ligase in the presence or absence of one or more synthetic double stranded oligonucleotides optionally containing cohesive ends.
- Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Sigma-Genosys Ltd, London Road, Pampisford, Cambridge, United Kingdom.
- The thio-albumin or fusions of thio-albumin and another protein or proteins may be expressed from a nucleotide sequence, which may or may not contain one or more introns. Additionally the nucleotide sequence may or may not be codon optimised for the host by methods known to the art.
- The thio-albumin or fusions of thio-albumin and another protein or proteins can be expressed as variants with reduced N-linked glycosylation. Accordingly, in case of human serum albumin (HSA), it may be particularly advantageous to use a yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins, for instance by disruption of the gene coding sequence. Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell. The mannosylated albumin would be able to bind to the lectin Concanavalin A. The amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may or may not be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced. Alternatively, the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody. Alternatively, the yeast could be cultured in the presence of a compound that inhibits the activity of one of the PMT genes (Duffy et al, “Inhibition of protein mannosyltransferase 1 (PMT1) activity in the pathogenic yeast Candida albicans”, International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, 26-31 August 2001, Monte Verita, Switzerland, Poster Abstract P38; the poster abstract may be viewed at http://www.micro.biol.ethz.ch/cellwall/). If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia pastoris or Kluyveromyces lactis. The sequence of PMT1 (or any other PMT gene) isolated from S. cerevisiae may be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species. The cloning of the PMT1 homologue of Kluyveromyces lactis is described in WO 94/04687.
- The yeast may or may not also have a deletion of the HSP150 and/or YAP3 genes as taught respectively in WO 95/33833 and WO 95/23857.
- The HSA variant may be produced by recombinant expression and secretion. Where the expression system (i.e. the host cell) is yeast, such as Saccharomyces cerevisiae, suitable promoters for S. cerevisiae include those associated with the PGK1 gene, GAL1 or GAL10 genes, TEF1, TEF2, PYK1, PMA1, CYC1, PHO5, TRP1, ADH1, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, a-mating factor pheromone, a-mating factor pheromone, the PRB1 promoter, the PRA1 promoter, the GPD1 promoter, and hybrid promoters involving hybrids of parts of 5′ regulatory regions with parts of 5′ regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).
- Suitable transcription termination signals are well known in the art. Where the host cell is eukaryotic, the transcription termination signal is preferably derived from the 3′ flanking sequence of a eukaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3′ flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different. In that case, and where the host is a yeast, preferably S. cerevisiae, then the termination signal of the S. cerevisiae ADH1, ADH2, CYC1, or PGK1 genes are preferred.
- It may be beneficial for the promoter and open reading frame of the gene encoding the recombinant protein comprising the sequence of an albumin mutant to be flanked by transcription termination sequences so that the transcription termination sequences are located both upstream and downstream of the promoter and open reading frame, in order to prevent transcriptional read-through into any neighbouring genes, such as 2 μm genes, and vice versa.
- In one embodiment, the favoured regulatory sequences in yeast, such as Saccharomyces cerevisiae, include: a yeast promoter (e.g. the Saccharomyces cerevisiae PRB1 promoter), as taught in
EP 431 880; and a transcription terminator, preferably the terminator from Saccharomyces ADH1, as taught inEP 60 057. - It may be beneficial for the non-coding region to incorporate more than one DNA sequence encoding a translational stop codon, such as UAA, UAG or UGA, in order to minimise translational read-through and thus avoid the production of elongated, non-natural fusion proteins. The translation stop codon UAA is preferred.
- In one preferred embodiment, the recombinant protein comprising the sequence of an albumin mutant is secreted. In that case, a sequence encoding a secretion leader sequence may be included in the open reading frame. Thus, a polynucleotide according to the present invention may comprise a sequence that encodes a recombinant protein comprising the sequence of an albumin mutant operably linked to a polynucleotide sequence that encodes a secretion leader sequence. Leader sequences are usually, although not necessarily, located at the N-terminus of the primary translation product of an ORF and are generally, although not necessarily, cleaved off the protein during the secretion process, to yield the “mature” protein. Thus, in one embodiment, the term “operably linked” in the context of leader sequences includes the meaning that the sequence that encodes a recombinant protein comprising the sequence of an albumin mutant is linked, at its 5′ end, and in-frame, to the 3′ end of a polynucleotide sequence that encodes a secretion leader sequence. Alternatively, the polynucleotide sequence that encodes a secretion leader sequence may be located, in-frame, within the coding sequence of the recombinant protein comprising the sequence of an albumin mutant, or at the 3′ end of the coding sequence of the recombinant protein comprising the sequence of an albumin mutant.
- Numerous natural or artificial polypeptide leader sequences (also called secretion pre regions and pre/pro regions) have been used or developed for secreting proteins from host cells. Leader sequences direct a nascent protein towards the machinery of the cell that exports proteins from the cell into the surrounding medium or, in some cases, into the periplasmic space.
- For production of proteins in eukaryotic species such as the yeasts Saccharomyces cerevisiae, Zygosaccharomyces species, Kluyveromyces lactis and Pichia pastoris, a secretion leader sequence may be used. This may comprise a signal (pre) sequence or a prepro leader sequence. Signal sequences are known to be heterogeneous in their amino acid sequence (Nothwehr and Gordon 1990,
Bioessays 12, 479-484, or Gierasch 1989,Biochemistry 28, p923-930). In essence, signal sequences are generally N-terminally located, have a basic n-region, a hydrophobic h-region and a polar c-region. As long as this structure is retained the signal sequence will work, irrespective of the amino acid composition. How well they work, i.e. how much mature protein is secreted, depends upon the amino acid sequence. Accordingly, the term “signal peptide” is understood to mean a presequence which is predominantly hydrophobic in nature and present as an N-terminal sequence of the precursor form of an extracellular protein expressed in yeast. The function of the signal peptide is to allow the expressed protein to be secreted to enter the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. Known leader sequences include those from the S. cerevisiae acid phosphatase protein (Pho5p) (seeEP 366 400), the invertase protein (Suc2p) (see Smith et aL (1985) Science, 229, 1219-1224) and heat-shock protein-150 (Hsp150p) (see WO 95/33833). Additionally, leader sequences from the S. cerevisiae mating factor alpha-1 protein (MFα-1) and from the human lysozyme and human serum albumin (HSA) protein have been used, the latter having been used especially, although not exclusively, for secreting human albumin. WO 90/01063 discloses a fusion of the MFα-1 and HSA leader sequences (also known as the fusion leader sequence (FL)). In addition, the natural albumin leader sequence may or may not be used to direct secretion of the recombinant protein comprising the sequence of an albumin mutant. - The skilled person will appreciate that any suitable plasmid may be used, such as a centromeric plasmid. The examples provide suitable plasmids (centromeric YCplac33-based vectors) for use to transform yeast host cells of the present invention. Alternatively, any other suitable plasmid may be used, such as a yeast-compatible 2pm-based plasmid.
- Plasmids obtained from one yeast type can be maintained in other yeast types (Irie et al, 1991, Gene, 108(1), 139-144; Irie et al, 1991, Mol. Gen. Genet., 225(2), 257-265). For example, pSR1 from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae. In one embodiment the plasmid may or may not be a 2 μm-family plasmid and the host cell will be compatible with the 2 μm-family plasmid used (see below for a full description of the following plasmids). For example, where the plasmid is based on pSR1, pSB3 or pSB4 then a suitable yeast cell is Zygosaccharomyces rouxii; where the plasmid is based on pSB1 or pSB2 then a suitable yeast cell is Zygosaccharomyces bailli; where the plasmid is based on pSM1 then a suitable yeast cell is Zygosaccharomyces fermentati; where the plasmid is based on pKD1 then a suitable yeast cell is Kluyveromyces drosophilarum; where the plasmid is based on pPM1 then a suitable yeast cell is Pichia membranaefaciens; where the plasmid is based on the 2 μm plasmid then a suitable yeast cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Thus, the plasmid may be based on the 2 μm plasmid and the yeast cell may be Saccharomyces cerevisiae. A 2 μm-family plasmid can be said to be “based on” a naturally occurring plasmid if it comprises one, two or preferably three of the genes FLP, REP1 and REP2 having sequences derived from that naturally occurring plasmid.
- Useful yeast episomal plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA), YEp24 (Botstein, D., et al. (1979)
Gene 8, 17-24), and YEplac122, YEplac195 and YEplac181 (Gietz, R. D. and Sugino. A. (1988)Gene 74, 527-534). Other yeast plasmids are described in WO 90/01063 andEP 424 117, as well as the “disintegration vectors of EP-A-286 424 and WO2005061719. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3, as are Ylplac204, Ylplac211 and Ylplac128 (Gietz, R. D. and Sugino. A. (1988)Gene 74, 527-534). Plasmids pRS413-416 are Yeast Centromere plasmids (YCps) as are YCplac22, YCplac33 and YCplac111 (Gietz, R. D. and Sugino. A. (1988)Gene 74, 527-534). - Where one or more of the helper (also known as ‘chaperone’) protein(s) and/or protein product of choice are encoded by a plasmid-borne polynucleotide sequence, the host cell type may be selected for compatibility with the plasmid type being used. Such plasmids are disclosed in WO2005061719. Preferred helper proteins include PDI1, AHAI, ATP11, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1, CPR3, CPRE, DER1, DER3, DOA4, ERO1, EUG1, ERV2, EPS1, FKB2, FMO1, HCH1, HRD3, HSP10, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, KAR2, JEM1, MDJ1, MDJ2, MPD1, MPD2, PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SCJ1, SSA1, SSA2, SSA3, SSA4, SSBI, SSB2, SSC1, SSE2, SIL1, SLS1, ORM1, ORM2, PERI, PTC2, PSE1, UBC7, UBI4 and HAC1 or a truncated intronless HAC1 (Valkonen et al. 2003, Applied Environ. Micro., 69, 2065). Such helper proteins are disclosed in WO 2005/061718, WO 2006/067511 and WO 2006/136831.
- Plasmids as defined herein may be introduced into a host through standard techniques. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.
Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978)Nature 275, 104-109 is also useful. Methods for the transformation of S. cerevisiae are taught generally inEP 251 744,EP 258 067 and WO 90/01063, all of which are incorporated herein by reference. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA. - Electroporation is also useful for transforming cells and is well known in the art for transforming fungal (including yeast) cell, plant cells, bacterial cells and animal (including vertebrate) cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
- Generally, the plasmid will transform not all of the hosts and it will therefore be necessary to select for transformed host cells. Thus, a plasmid may comprise a selectable marker, including but not limited to bacterial selectable marker and/or a yeast selectable marker. A typical bacterial selectable marker is the β-lactamase gene although many others are known in the art. Typical yeast selectable marker include LEU2, TRP1, HIS3, HIS4, URA3, URA5,
SFA 1, ADE2, MET15, LYS5, LYS2, ILV2, FBA1, PSE1, PDI1 and PGK1. Those skilled in the art will appreciate that any gene whose chromosomal deletion or inactivation results in an unviable host, so called essential genes, can be used as a selective marker if a functional gene is provided on the plasmid, as demonstrated for PGK1 in a pgk1 yeast strain (Piper and Curran, 1990, Curr. Genet. 17, 119). Suitable essential genes can be found within the Stanford Genome Database (SGD), (http:://db.yeastgenome.org). Any essential gene product (e.g. PDI1, PSE1, PGK1 or FBA1) which, when deleted or inactivated, does not result in an auxotrophic (biosynthetic) requirement, can be used as a selectable marker on a plasmid in a host cell that, in the absence of the plasmid, is unable to produce that gene product, to achieve increased plasmid stability without the disadvantage of requiring the cell to be cultured under specific selective conditions. By “auxotrophic (biosynthetic) requirement” we include a deficiency which can be complemented by additions or modifications to the growth medium. Therefore, preferred “essential marker genes” in the context of the present application are those that, when deleted or inactivated in a host cell, result in a deficiency which cannot be complemented by additions or modifications to the growth medium. Additionally, a plasmid may comprise more than one selectable marker. - Transformed host cells may be cultured for a sufficient time and under appropriate conditions known to those skilled in the art, and in view of the teachings disclosed herein, to permit the expression of the helper protein(s) and the protein product of choice.
- The culture medium may be non-selective or place a selective pressure on the maintenance of a plasmid.
- Methods for culturing prokaryotic host cells, such as E. coli, and eukaryotic host cells, such as mammalian cells are well known in the art. Methods for culturing yeast are generally taught in
EP 330 451 andEP 361 991. - The thus produced protein product of choice may be present intracellularly or, if secreted, in the culture medium and/or periplasmic space of the host cell.
- The step of “purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium” optionally comprises cell immobilisation, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilisation, separation and/or breakage.
- Thio-albumin of the invention may be purified from the culture medium by any technique that has been found to be useful for purifying such proteins. Similarly, cell separation techniques, such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like) are well known in the art. Likewise, methods of cell breakage, including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
- The “at least one other purification step” may be any other step suitable for protein purification known in the art. For example purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye;
EP 464 590, removal of yeast-derived colorants;EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference. Suitable methods include ammonium sulphate or ethanol precipitation, acid or solvent extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography (“HPLC”), reverse phase HPLC, conductivity adjustment and the like. - The polypeptide may be purified to a commercially or industrially acceptable level of purity. By commercially or industrially acceptable level of purity, we include the provision of the thio-albumin and/or thio-albumin-conjugate in which other material (for example, one or more contaminants) are present at a level of less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% and, most preferably at a level of 0%.
- A commercially or industrially acceptable level of purity may be obtained by a relatively crude purification method by which the protein product of choice is put into a form suitable for its intended purpose. A protein preparation that has been purified to a commercially or industrially acceptable level of purity may, in addition to the protein product of choice, also comprise, for example, cell culture components such as host cells or debris derived therefrom. Alternatively, high molecular weight components (such as host cells or debris derived therefrom) may or may not be removed (such as by filtration or centrifugation) to obtain a composition comprising the protein product of choice and, optionally, a functionally acceptable level of low molecular weight contaminants derived from the cell culture process.
- The protein may or may not be purified to achieve a pharmaceutically acceptable level of purity. A protein has a pharmaceutically acceptable level of purity if it is essentially pyrogen free and can be used for its intended purpose and hence be administered in a pharmaceutically efficacious amount without causing medical effects not associated with the activity of the protein.
- The thio-albumin and/or thio-albumin-conjugate may be provided at a concentration of at least 10−4 g.L−1, 10−3 g.L−1, 0.01 g.L−1, 0.02 g.L−1, 0.03 g.L−1, 0.04 g.L−1, 0.05 g.L−1, 0.06 g.L−1, 0.07 g.L−1, 0.08 g.L−1, 0.09 g.L−1, 0.1 g.L−1, 0.2 g.L−l, 0.3 g.L−1, 0.4 g.L−1, 0.5 g.L−1, 0.6 g/L−1, 0.7 g.L−1, 0.8 g.L−1, 0.9 0 g.L−1, 1 g.L−1, 2 g.L−1, 3 g.L−1, 4 g.L−1, 5 g.L−1, 6 g.L−1, 7 g.L−1, 8 g.L−1, 9 g.L−1, 10 g.L−1, 15 g.L−1, 20 g.L−1, 25 g.L−1, 30 g.L−1, 40 g.L−1, 50 g.L−1, 60 g.L−1, 70 g.L−1, 70 g.L−1, 90 gL−1, 100 g.L−1, 150 g.L−1, 200 g.L−1, 250 g.L−1, 300 g.L−1, 350 g.L−1, 400 g.L−1, 500 g.L−1, 600 g.L−1, 700 g.L−1, 800 g.L−1, 900 g.L−1, 1000 g.L−1.
- A method of the present invention may or may not further comprise the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
- Although it is possible for a therapeutically useful protein obtained by a process of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers or diluents. The carrier(s) or diluent(s) must be “acceptable” in the sense of being compatible with the desired protein. Typically, the carriers or diluents will be water or saline which will be sterile and pyrogen free.
- Alternatively, a method of the present invention may or may not further comprise the step of lyophilising the thus purified protein product of choice.
- Formulation of Thio-Albumin or Conjugate
- The thio-albumin may be formulated by strategies given in “Protein Formulation and Delivery”, E. J. McNally (Ed.), published by Marcel Dekker Inc.
New York 2000 and “Rational Design of Stable Protein Formulations-Theory and Practice”; J. F. Carpenter and M. C. Manning (Ed.)Pharmaceutical Biotechnology Vol 13. Kluwer Academic/Plenum Publishers, New York 2002, Yazdi and Murphy, (1994)Cancer Research 54, 6387-6394, Widera et al., (2003)Pharmaceutical Research 20, 1231-1238; Lee et al., (2005) Arch. Pharm. Res. 28, 722-729. Examples of formulation methods are as follows: - Method #1: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., −20° C. or −80° C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl.
- Method #2: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., −20° C. or −80° C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl and containing 10-20 mg/
L Polysorbate 80. - Method #3: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., −20° C. or −80° C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0).
- Method #4: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4° C., −20° C. or −80° C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0) containing 10-20 mg/
L Polysorbate 80. - Method #5: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against water, freeze dried and stored at 4° C., −20° C. or −80° C.
- Method #6: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against 0.01 M-0.2 M NaCl (pH 7.0-8.0), freeze dried and stored at 4° C., −20° C. or −80° C.
- The thio-albumin of the invention (and/or its conjugated form) may be used to produce nanoparticles and/or be entrapped within a nanoparticle or liposome.
- The thio-albumin of the invention may be used with and/or in and/or as a nanoparticle and/or liposome. A problem of current conjugation strategies is maintaining both the pharmacological and immunological activity of the conjugation partner, such as a bioactive-targeting ligand conjugate. There is likely to be a maximum number of protein targeting ligand/bioactive moieties (conjugation partners) possible for conjugation to a protein and if this number is exceeded the targeting ligand does not retain its biological activity. Preferably the biological activity of the conjugation partner is not reduced by conjugation to an albumin of the invention.
- Liposomes and nanoparticles may be used to entrap bioactive compounds. They provide a mechanism for enhanced delivery of drugs such as bioactive compounds, or uptake by target cells and/or a reduction in the toxicity of the free bioactive to non-target organs which may result in an increased therapeutic index and/or reduced side effects. In addition, many solvent-based formulations required for the delivery of some bioactive compounds (e.g. taxanes) are associated with toxicity which limits the maximum dose which can be given to a patient. Liposome and nanoparticle delivery may also be advantageous for such bioactive compounds, since they would allow larger amounts of the bioactive compound to be delivered whilst avoiding some of the toxicities of solvent-based formulations (Hawkins et al (2008) Advanced Drug Delivery Reviews, 60, 8, p 876-885).
- Methods for attaching targeting ligands to liposomes and nanoparticles are known in the art (reviewed in Nobs et al (2004) Journal of Pharmaceutical Sciences Vol 93 p 980-1992) and may be used in accordance with the invention. Attachment methods may be non-covalent or covalent. Covalent reactions appear to be favourable, because covalent linkage is more stable than noncovalent methods. Lipids for the covalent or non-covalent attachment of proteins, peptides, or drugs to the liposome surface are available commercially (for example Avanti Polar Lipids Inc Alabaster, Ala., USA). There are 3 major classes of functionality: conjugation through disulphide or thioether formation, amide bond formation, or biotin/streptavidin binding, any of these may be used in the invention.
- A number of methods relying on covalent coupling ligands to the surface of liposomes via thioether bonds have been described, most commonly utilizing the highly efficient reaction of maleimide with thiol groups. Functionalized lipid anchors commonly added to liposomes, and which may be used in or with the invention, include, but are not limited those containing maleimide such as N-[4-(p-maleimidophenyl) butyramide]-PE (N-MPB]-PE) or N-[4-(p-maleimidomethyl) cyclohexane-carboxamide) (MCC-PE) which allow convenient covalent coupling of the targeting moiety via a stable thioether bond (Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288).
- Method #7: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be formulated into nanoparticles prepared according to known procedures for preparing nanoparticles, such as procedures disclosed in WO 2004/071536 A1 and WO 2008/007146 A1, both incorporated herein by reference.
- Similarly materials for the formation of nanoparticles, including but are limited to Poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and COOH-PLA are commercially available and may be functionalized with maleimide or other known chemistries according to known literature for nanoparticle formation. Any of these may be used in or with the invention.
- Another convenient way for covalent coupling of ligands to liposomes involves conjugation of two thiols to form a disulphide; however under the reductive conditions in serum more stable conjugation chemistries involving one free thiol group may be preferred. Chemistries such as (PDP-PE) allow covalent coupling via a disulphide bond. Modification of the ligand to introduce a free thiol group or a functionalized linker may be used. An advantage of the thio-albumin of the invention is that no ligand modification is required. However, ligand modification may optionally be used in addition to the invention.
- Frequently thiol groups are not present in proteins, or are not present in sufficient amounts or at the desired location. Thus, most cases of covalent coupling of one of more ligands to a liposome via thioether or disulphide bonds requires the use of heterobifunctional cross linking agents (described herein with reference to conjugation). Some heterobifunctional cross linking agents (such as SPDP and SATA) require a de-protection step. The thio-albumin of the invention overcomes the requirement for this additional processing.
- Alternatively thio-albumin could be conjugated to liposomes or nanoparticles by other chemistries, known to the art. For example, thio-albumin could be attached by an amide bond using a functionalised lipid anchor with either amine or carboxyl functional groups (examples include DSPE-PEG-COOH) which reacts with the primary amine of the ligand. Direct cross linking between primary amines and the surface of liposomes may also be used. The one or more free thiol groups of thio-albumin would then be available for conjugation to another conjugation partner.
- Following conjugation, a conjugation partner (e.g. bioactive molecule) may show a reduction in its activity (e.g. bioactivity). Thio-albumin described in this invention may overcome this problem by providing a conjugate, nanoparticle and/or liposome in which the conjugation partner is located and/or orientated with respect to a thio-albumin such that the conjugation partner retains at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of its unconjugated activity.
- The term ‘conjugation partner’ includes bioactive agents, imaging agents, diagnostic agents, contrast agents and therapeutic compounds such as chemotherapeutic drugs and radiopharmaceuticals. A thio-albumin of the invention may be conjugated to one or more conjugation partners.
- The use of diagnostic agents, imaging agents and biological “contrast” agents are well known to the art. A diagnostic agent is any pharmaceutical product used as part of a diagnostic test (i.e. together with the equipment and procedures that are needed to assess the test result). The diagnostic agent may be used in vivo, ex vivo or in vitro.
- The ability of albumin to accumulate in damaged muscle fibres of dystrophic muscle has been well described. For example, a Gadolinium-DTPA-albumin conjugate may be used as a combined diagnostic and therapeutic tool to visualize and monitor, for example, dystrophic muscle by magnetic resonance imaging (MRI) and for the delivery of putative therapeutics bound to albumin for effective targeting to dystrophic muscle (Amthor et al. (2004) Neuromuscular Disorders 14912: 791-796). Malignant tumours often show an increased uptake and metabolism of albumin. The use of gadolinium-albumin conjugate has also been described for improved imaging of malignant tumours and to determine by MRI tumours sensitive to a therapy with drug-conjugated albumin (Kiessling et al. (2002) Investigative Radiology 37(4): 93-198).
- Current imaging agents often degrade quickly whilst longer-lasting agents are often toxic. The use of albumin conjugates may be especially useful to increase the half life of imaging agents and would therefore permit imaging over an extended period of time. WO2005/082423 describes the use of serum albumin conjugated to fluorescent substances for imaging.
- A thio-albumin of this invention may be conjugated to two or more molecules selected from imaging agents, diagnostic agents, therapeutic compounds and contrast agents.
- Tumours (and muscle degeneration) show enhanced uptake of albumin (EPR: Enhanced Permeation and Retention). Albumin conjugates may be used for enhanced imaging, and also to assess whether tumours (or or other tissues and organs) would be suitable for albumin conjugated drugs.
- The bioactive compound may be a therapeutic or diagnostic compound. The therapeutic compound may be a chemotherapy drug for use in cancer chemotherapy. It may be cytostatic or cytotoxic; it may be a tumor-inhibiting agent.
- The bioactive compound may already contain a free thiol group, e.g. a polypeptide containing a Cysteine residue with a free thiol group. Alternatively, the bioactive compound may be modified so as to contain a free thiol group. Thus, the amino acid sequence of a polypeptide may be altered so as to include a Cysteine residue with a free thiol group, or the bioactive compound may be chemically derivatized to include a free thiol group.
- The bioactive compound may be a polypeptide (protein), particularly a recombinant protein pharmaceutical. It may be a chemotherapy or radiotherapy drug used to treat cancers and other related diseases.
- The free thiol containing albumin mutein of the invention (thio-albumin) can be conjugated via the free thiol group, or groups if the albumin mutein of the invention contains more than one free thiol, to at least one bioactive compound by methods know to the art. The bioactive compound includes but is not limited to, peptides, polypeptides or proteins (either natural, recombinant, or synthetic) (Debinski, (2002)
Cancer Investigation 20, 801-809, O'Keefe and Draper et al., (1985)JBC 260, 932-937, Xia et al., (2000) J.Pharmacology Experimental Therapeutics 295, 594-600, Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384, Humphries, et al., (1994) J.Tissue Culture Methods 16, 239-242, Wenning et al., (1998) Biotech. Bioeng. 57, 484-496, Yazdi and Murphy, (1994)Cancer Research 54, 6387-6394, Weaver and Laske (2003) J. Neuro-Oncology 65, 3-13, Widera at al., (2003)Pharmaceutical Research 20, 1231-1238, Daniels, T. R. et al. (2006)Clinical Immunology 121, 159-176 and the references included therein); therapeutic and diagnostic drugs or compounds (Mishra et al., (2006) J. Drug Targeting 14, 45-53, Lim and Shen, (2004)Pharmaceutical Research 21, 1985-1992, Fritzer et al., (1996)Biochemical Pharmacology 51, 489-493, Lubgan and Jozwiak (2002) Cell. Mol. Biol. Lett. 7, 98, Daniels, T. R. at al. (2006)Clinical Immunology 121, 159-176 and the references included therein); high molecular weight complexes including but not limited to liposomes, viruses and nanoparticles (Mishra at al., (2006) J. Drug Targeting 14, 45-53, Daniels, T. R. at al. (2006)Clinical Immunology 121, 159-176 and the references included therein); nucleic acids and radionuclides, including DNA, RNA (including siRNA) and their analogs (Lee at al., (2005) Arch. Pharm. Res. 28, 722-729, Huang et al., (2007) FASEB J. 21, 1117-1125, Daniels, T. R. et al. (2006)Clinical Immunology 121, 159-176 and the references included therein) and devices (Humphries, et al., (1994) J.Tissue Culture Methods 16, 239-242 and the references included therein). Additionally the entity can itself be modified by methods known to the art. - 4-1BB ligand, 5-helix, A human C-C chemokine, A human L105 chemokine, A human L105 chemokine designated huL105_3., A monokine induced by gamma-interferon (MIG), A partial CXCR4B protein, A platelet basic protein (PBP), α1-antitrypsin, □□ACRP-30 Homologue; Complement Component C1q C, Adenoid-expressed chemokine (ADEC), aFGF; FGF-1, AGF, AGF Protein, albumin, an etoposide, angiostatin, Anthrax vaccine, Antibodies specific for collapsin, antistasin, Anti-TGF beta family antibodies, antithrombin APM-1; ACRP-30; Famoxin, apo-lipoprotein species, Arylsulfatase B, b57 Protein, BCMA, Beta-thromboglobulin protein (beta-TG), bFGF; FGF2, Blood coagulation factors, BMP Processing Enzyme Furin, BMP-10, BMP-12, BMP-15, BMP-17, BMP-18, BMP-2B, BMP-4, BMP-5, BMP-6, BMP-9, Bone Morphogenic Protein-2, calcitonin, Calpain-10a, Calpain-10b, Calpain-10c, Cancer Vaccine, Carboxypeptidase, C-C chemokine, MCP2, CCR5 variant, CCR7, CCR7, CD11a Mab, CD137; 4-1BB Receptor Protein, CD20 Mab, CD27, CD27L, CD30, CD30 ligand, CD33 immunotoxin, CD40, CD40L, CD52 Mab, Cerebus Protein, Chemokine Eotaxin., Chemokine hIL-8, Chemokine hMCP1, Chemokine hMCP1a, Chemokine hMCP1b, Chemokine hMCP2, Chemokine hMCP3, Chemokine hSDF1b, Chemokine MCP-4, chemokine TECK and TECK variant, Chemokine-like protein IL-8M1 Full-Length and Mature, Chemokine-like protein IL-8M10 Full-Length and Mature, Chemokine-like protein IL-8M3, Chemokine-like protein IL-8M8 Full-Length and Mature, Chemokine-like protein IL-8M9 Full-Length and Mature, Chemokine-like protein PF4-414 Full-Length and Mature, Chemokine-like protein PF4-426 Full-Length and Mature, Chemokine-like protein PF4-M2 Full-Length and Mature, Cholera vaccine, Chondromodulin-like protein, c-kit ligand; SCF; Mast cell growth factor; MGF; Fibrosarcoma-derived stem cell factor, CNTF and fragment thereof (such as CNTFAx15′(Axokine™)), coagulation factors in both pre and active forms, collagens, Complement C5 Mab, Connective tissue activating protein-Ill, CTAA16.88 Mab, CTAP-III, CTLA4-Ig, CTLA-8, CXC3, CXC3, CXCR3; CXC chemokine receptor 3, cyanovirin-N, Darbepoetin, designated exodus, designated huL105_7., DIL-40, Dnase, EDAR, EGF Receptor Mab, ENA-78, Endostatin, Eotaxin, Epithelial neutrophil activating protein-78, EPO receptor; EPOR, erythropoietin (EPO) and EPO mimics, Eutropin, Exodus protein, Factor IX, Factor VII, Factor VIII, Factor X and Factor XIII, FAS Ligand Inhibitory Protein (DcR3), FasL, FasL, FasL, FGF, FGF-12; Fibroblast growth factor homologous factor-1, FGF-15, FGF-16, FGF-18, FGF-3; INT-2, FGF-4; gelonin, HST-1; HBGF-4, FGF-5, FGF-6; Heparin binding secreted transforming factor-2, FGF-8, FGF-9; Glia activating factor, fibrinogen, flt-1, flt-3 ligand, Follicle stimulating hormone Alpha subunit, Follicle stimulating hormone Beta subunit, Follitropin, Fractalkine, fragment. myofibrillar protein Troponin I, FSH, Galactosidase, Galectin-4, G-CSF, GDF-1, Gene therapy, Glioma-derived growth factor, glucagon, glucagon-like peptides, Glucocerebrosidase, glucose oxidase, Glucosidase, Glycodelin-A; Progesterone-associated endometrial protein, GM-CSF, gonadotropin, Granulocyte chemotactic protein-2 (GCP-2), Granulocyte-macrophage colony stimulating factor, growth hormone, Growth related oncogene-alpha (GRO-alpha), Growth related oncogene-beta (GRO-beta), Growth related oncogene-gamma (GRO-gamma), hAPO-4; TROY, hCG, Hepatitus B surface Antigen, Hepatitus B Vaccine, HER2 Receptor Mab, hirudin, HIV gp120, HIV gp41, HIV Inhibitor Peptide, HIV Inhibitor Peptide, HIV Inhibitor Peptide, HIV protease inhibiting peptides, HIV-1 protease inhibitors, HPV vaccine, Human 6CKine protein, Human Act-2 protein, Human adipogenesis inhibitory factor, human B cell stimulating factor-2 receptor, Human beta-chemokine H1305 (MCP-2), Human C-C chemokine DGWCC, Human CC chemokine ELC protein, Human CC type chemokine interleukin C, Human CCC3 protein, Human CCF18 chemokine, Human CC-type chemokine protein designated SLC (secondary lymphoid chemokine), Human chemokine beta-8 short forms, Human chemokine 010, Human chemokine CC-2, Human chemokine CC-3, Human chemokine CCR-2, Human chemokine Ckbeta-7, Human chemokine ENA-78, Human chemokine eotaxin, Human chemokine GRO alpha, Human chemokine GROalpha, Human chemokine GRObeta, Human chemokine HCC-1, Human chemokine HCC-1, Human chemokine I-309, Human chemokine IP-10, Human chemokine L105_3, Human chemokine L105_7, Human chemokine MIG, Human chemokine MIG-beta protein, Human chemokine MIP-1alpha, Human chemokine MIP1beta, Human chemokine MIP-3alpha, Human chemokine MIP-3beta, Human chemokine PF4, Human chemokine protein 331D5, Human chemokine protein 61164, Human chemokine receptor CXCR3, Human chemokine SDF1alpha, Human chemokine SDF1beta, Human chemokine ZSIG-35, Human Chr19Kine protein, Human CKbeta-9, Human CKbeta-9, Human CX3C 111 amino acid chemokine, Human DNAX interleukin-40, Human DVic-1 C-C chemokine, Human EDIRF I protein sequence, Human EDIRF II protein sequence, Human eosinocyte CC type chemokine eotaxin, Human eosinophil-expressed chemokine (EEC), Human fast twitch skeletal muscle troponin C, Human fast twitch skeletal muscle troponin I, Human fast twitch skeletal muscle Troponin subunit C, Human fast twitch skeletal muscle Troponin subunit I Protein, Human fast twitch skeletal muscle Troponin subunit T, Human fast twitch skeletal muscle troponin T, Human foetal spleen expressed chemokine, FSEC, Human GM-CSF receptor, Human gro-alpha chemokine, Human gro-beta chemokine, Human gro-gamma chemokine, Human IL-16 protein, Human IL-1RD10 protein sequence, Human IL-1RD9, Human IL-5 receptor alpha chain, Human IL-6 receptor, Human IL-8 receptor protein hIL8RA, Human IL-8 receptor protein hIL8RB, Human IL-9 receptor protein, Human IL-9 receptor protein variant #3, Human IL-9 receptor protein variant fragment, Human IL-9 receptor protein variant fragment#3, Human interleukin 1 delta, Human Interleukin 10, Human Interleukin 10, Human interleukin 18, Human interleukin 18 derivatives, Human interleukin-1 beta precursor, Human interleukin-1 beta precursor., Human interleukin-1 receptor accessory protein, Human interleukin-1 receptor antagonist beta, Human interleukin-1 type-3 receptor, Human Interleukin-10 (precursor), Human Interleukin-10 (precursor), Human interleukin-11 receptor, Human interleukin-12 40 kD subunit, Human interleukin-12 beta-1 receptor, Human interleukin-12 beta-2 receptor, Human Interleukin-12 p35 protein, Human Interleukin-12 p40 protein, Human interleukin-12 receptor, Human interleukin-13 alpha receptor, Human interleukin-13 beta receptor, Human interleukin-15, Human interleukin-15 receptor from clone P1, Human interleukin-17 receptor, Human interleukin-18 protein (IL-18), Human interleukin-3, human interleukin-3 receptor, Human interleukin-3 variant, Human interleukin-4 receptor, Human interleukin-5, Human interleukin-6, Human interleukin-7, Human interleukin-7., Human interleukin-8 (IL-8), Human intracellular IL-1 receptor antagonist, Human IP-10 and HIV-1 gp120 hypervariable region fusion protein, Human IP-10 and human Muc-1 core epitope (VNT) fusion protein, human liver and activation regulated chemokine (LARC), Human Lkn-1 Full-Length and Mature protein, Human mammary associated chemokine (MACK) protein Full-Length and Mature, Human mature chemokine Ckbeta-7, Human mature gro-alpha, Human mature gro-gamma polypeptide used to treat sepsis, Human MCP-3 and human Muc-1 core epitope (VNT) fusion protein, Human MI10 protein, Human MI1A protein, Human monocyte chemoattractant factor hMCP-1, Human monocyte chemoattractant factor hMCP-3, Human monocyte chemotactic proprotein (MCPP) sequence, Human neurotactin chemokine like domain, Human non-ELR CXC chemokine H174, Human non-ELR CXC chemokine IP10, Human non-ELR CXC chemokine Mig, Human PAI-1 mutants, Human protein with IL-16 activity, Human protein with IL-16 activity, Human secondary lymphoid chemokine (SLC), Human SISD protein, Human STCP-1, Human stromal cell-derived chemokine, SDF-1, Human T cell mixed lymphocyte reaction expressed chemokine (TMEC), Human thymus and activation regulated cytokine (TARC), Human thymus expressed, Human TNF-alpha, Human TNF-alpha, Human TNF-beta (LT-alpha), Human type CC chemokine eotaxin 3 protein sequence, Human type II interleukin-1 receptor, Human wild-type interleukin-4 (hIL-4) protein, Human ZCHEMO-8 protein, Humanized Anti-VEGF Antibodies, and fragments thereof, Humanized Anti-VEGF Antibodies, and fragments thereof, Hyaluronidase, ICE 10 kD subunit., ICE 20 kD subunit., ICE 22 kD subunit., Iduronate-2-sulfatase, Iduronidase, IL-1 alpha, IL-1 beta, IL-1 inhibitor (IL-10., IL-1 mature, IL-10 receptor, IL-11, IL-11, IL-12 p40 subunit., IL-13, IL-14, IL-15, IL-15 receptor, IL-17, IL-17 receptor, II-17 receptor, II-17 receptor, IL-19, IL-1i fragments, IL1-receptor antagonist, IL-21 (TIF), IL-3 containing fusion protein., IL-3 mutant proteins, IL-3 variants, IL-3 variants, IL-4, IL-4 mutein, IL-4 mutein Y124G, IL-4 mutein Y124X, IL-4 muteins, II-5 receptor, IL-6, II-6 receptor, IL-7 receptor clone, IL-8 receptor, IL-9 mature protein variant (Met117 version), immunoglobulins or immunoglobulin-based molecules or fragment of either (e.g. a Small Modular ImmunoPharmaceutical™ (“SMIP”) or dAb, Fab′ fragments, F(ab′)2, scAb, scFv or scFv fragment), including but not limited to plasminogen, Influenza Vaccine, Inhibin alpha, Inhibin beta, insulin, insulin-like growth factor, Integrin Mab, inter-alpha trypsin inhibitor, inter-alpha trypsin inhibitor, Interferon gamma-inducible protein (IP-10), interferons (such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species), interferons (such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species), Interleukin 6, Interleukin 8 (IL-8) receptor, Interleukin 8 receptor B, Interleukin-1alpha, Interleukin-2 receptor associated protein p43, interleukin-3, interleukin-4 muteins, Interleukin-8 (IL-8) protein., interleukin-9, Interleukin-9 (IL-9) mature protein (Thr117 version), interleukins (such as IL10, IL11 and IL2), interleukins (such as IL10, IL11 and IL2), Japanese encephalitis vaccine, Kalikrein Inhibitor, Keratinocyte growth factor, Kunitz domain protein (such as aprotinin, amyloid precursor protein and those described in WO 03/066824, with or without albumin fusions), Kunitz domain protein (such as aprotinin, amyloid precursor protein and those described in WO 03/066824, with or without albumin fusions), LACI, lactoferrin, Latent TGF-beta binding protein II, leptin, Liver expressed chemokine-1 (LVEC-1), Liver expressed chemokine-2 (LVEC-2), LT-alpha, LT-beta, Luteinization Hormone, Lyme Vaccine, Lymphotactin, Macrophage derived chemokine analogue MDC (n+1), Macrophage derived chemokine analogue MDC-eyfy, Macrophage derived chemokine analogue MDC-yl, Macrophage derived chemokine, MDC, Macrophage-derived chemokine (MDC), Maspin; Protease Inhibitor 5, MCP-1 receptor, MCP-1a, MCP-1b, MCP-3, MCP-4 receptor, M-CSF, Melanoma inhibiting protein, Membrane-bound proteins, Met117 human interleukin 9, MIP-3 alpha, MIP-3 beta, MIP-Gamma, MIRAP, Modified Rantes, monoclonal antibody, MP52, Mutant Interleukin 6 S176R, myofibrillar contractile protein Troponin I, Natriuretic Peptide, Nerve Growth Factor-beta, Nerve Growth Factor-beta2, Neuropilin-1, Neuropilin-2, Neurotactin, Neurotrophin-3, Neurotrophin-4, Neurotrophin-4a, Neurotrophin-4b, Neurotrophin-4c, Neurotrophin-4d, Neutrophil activating peptide-2 (NAP-2), NOGO-66 Receptor, NOGO-A, NOGO-B, NOGO-C, Novel beta-chemokine designated PTEC, N-terminal modified chemokine GroHEK/hSDF-1alpha, N-terminal modified chemokine GroHEK/hSDF-1 beta., N-terminal modified chemokine met-hSDF-1 alpha, N-terminal modified chemokine met-hSDF-1 beta, OPGL, Osteogenic Protein-1; OP-1; BMP-7, Osteogenic Protein-2, OX40; ACT-4, OX40L, Oxytocin (Neurophysin I), parathyroid hormone, Patched, Patched-2, PDGF-D, Pertussis toxoid, Pituitary expressed chemokine (PGEC), Placental Growth Factor, Placental Growth Factor-2, Plasminogen Activator Inhibitor-1; PAI-1, Plasminogen Activator Inhibitor-2; PAI-2, Plasminogen Activator Inhibitor-2; PAI-2, Platelet derived growth factor, Platelet derived growth factor Bv-sis, Platelet derived growth factor precursor A, Platelet derived growth factor precursor B, Platelet Mab, platelet-derived endothelial cell growth factor (PD-ECGF), Platelet-Derived Growth Factor A chain, Platelet-Derived Growth Factor B chain, polypeptide used to treat sepsis, Preproapolipoprotein “milano” variant, Preproapolipoprotein “paris” variant, pre-thrombin, Primate CC chemokine “ILINCK”, Primate CXC chemokine “IBICK”, proinsulin, Prolactin, Prolactin2, prosaptide, Protease inhibitor peptides, Protein C, Protein S, pro-thrombin, prourokinase, RANTES, RANTES 8-68, RANTES 9-68, RANTES peptide, RANTES receptor, Recombinant interleukin-16, Resistin, restrictocin, Retroviral protease inhibitors, ricin, Rotavirus Vaccine, RSV Mab, saporin, sarcin, Secreted and Transmembrane polypeptides, Secreted and Transmembrane polypeptides, serum cholinesterase, serum protein (such as a blood clotting factor), Soluble BMP Receptor Kinase Protein-3, Soluble VEGF Receptor, Stem Cell Inhibitory Factor, Straphylococcus Vaccine, Stromal Derived Factor-1 alpha, Stromal Derived Factor-1 beta, Substance P (tachykinin), T1249 peptide, T20 peptide, T4 Endonuclease, TACI, Tarc, TGF-beta 1, TGF-beta 2, Thr117 human interleukin 9, thrombin, Thrombopoietin derivativel, Thrombopoietin derivative2, Thrombopoietin derivative3, Thrombopoietin derivative4, Thrombopoietin derivative5, Thrombopoietin derivative6, Thrombopoietin derivative7, Thymus expressed chemokine (TECK), Thyroid stimulating Hormone, tick anticoagulant peptide, Tim-1 protein, TNF-alpha precursor, TNF-R, TNF-RII; TNF p75 Receptor; Death Receptor, tPA, transferrin, transforming growth factor beta, Troponin peptides, Truncated monocyte chemotactic protein 2 (6-76), Truncated monocyte chemotactic protein 2 (6-76), Truncated RANTES protein (3-68), tumour necrosis factor, Urate Oxidase, urokinase, Vasopressin (Neurophysin II), VEGF R-3; flt-4, VEGF Receptor; KDR; flk-1, VEGF-110, VEGF-121, VEGF-138, VEGF-145, VEGF-162, VEGF-165, VEGF-182, VEGF-189, VEGF-206, VEGF-D, VEGF-E; VEGF-X, von Willebrand's factor, Wild type monocyte chemotactic protein 2, Wild type monocyte chemotactic protein 2, ZTGF-beta 9.
- 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, A, Abraxane, Accutane®, Actinomycin-D, Adriamycin®, Adrucil®, Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole, Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine, BCG, BCNU, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225 , Calcium Leucovorin, Campeth®, Camptosar®, Camptothecin-11, Capecitabine, Carac™, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®, Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, Denileukin diftitox, DepoCyt™, Dexamethasone, Dexamethasone acetate , Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin liposomal, Droxia™, DTIC, DTIC-Dome®, Duralone®, Efudex®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®, Epirubicin, Epoetin alfa, Erbitux™, Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide Phosphate, Eulexin®, Evista®, Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Floxuridine, Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar®, Gleevec™, Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte-Colony Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexalen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin®, Idarubicin, Ifex®, IFN-alpha , Ifosfamide, IL-11 , IL-2 , Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin, Kidrolase®, Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine™, Leuprolide, Leurocristine, Leustatin™, Liposomal Ara-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, M, Matulane®, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®, Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex™, Methotrexate, Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine , Mutamycin®, Myleran®, Mylocel™, Mylotarg®, Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®, Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with Carmustine Implant, Purinethol®, R, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®, VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, Zevalin™, Zinecard®, Zoladex®, Zoledronic acid, Zolinza, Zometa®.
- Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Erbium-169, Fluorine-18, Gallium-67, Gold-198, Indium-111, Indium-113m, Iodine-123, Iodine-125, Iodine-131, Iron-59, Krypton-81m, Nitrogen-13, Oxygen-15, Phosphorous-32, Rhenium-186, Rubidium-82, Samarium-153, Selenium-75, Strontium-89, Technetium-99m, Thallium-201, Tritium, Xenon-127, Xenon-133, Yttrium-90.
- Gadolinium, magnetite, manganese, technetium, I125, I131, P32, TI201, Iopamidol, PET-FDG.
- The albumin may also be fused to one or more purification tags such as (Ala-Trp-Trp-Pro)n, avidin/streptavidin/Strep-tag, BCCP, B-tag (VP7 protein region of bluetongue virus), calmodulin binding protein (CBP), cellulose binding domains (CBD's), chitin binding domain, chloramphenicol acetyltransferase, c-myc, dihydrofolate reductase (DHFR), FLAG™ peptide (DYKDDDDK), galactose-binding protein, glutathione-S-transferase (GST), green flourescent protein (GFP), Growth hormone, N-terminus, hemagglutinin influenza virus (HAI), His-patch thioredoxin, His-tag, HSB-tag, KSI, lacZ (β-Galactosidase), maltose binding protein (MBP), NusA, ompT/ompA/pelB/DsbA/DsbC, polyarginine, polyaspartic acid, polycysteine, polyphenyalanine, S-tag, staphylococcal protein A, streptococcal protein G, T4 gp55, T7gene10, T7-tag, thioredoxin, trpE, ubiquitin.
- HSA has ligand binding and esterase activities, as described in “All about Albumin”, T. Peters Jr., Academic Press N.Y. The ligand binding properties include binding to anionic and neutral ligands such as long-chain fatty acids, bilirubin and other miscellaneous ligands. The long-chain fatty acids, oleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and palmitoleic (C16:1) are known to bind HSA.
- The polypeptide may include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially reduce the useful ligand-binding, immunological or receptor binding properties of albumin, for example to FcRN, bilirubin and/or a fatty acid. The polypeptide may have at least 5%, 10%, 15%, 20%, 30%, 40% or 50%, 60%, 70%, at least 80%, 90%, 95%, 100%, 105% or more of human serum albumin's receptor binding activity, mole for mole. The polypeptide may have increased affinity for an albumin receptor.
- Ligand binding studies can be performed on HSA and thio-albumins using an isothermal titration calorimetry method that had been suitably qualified for this purpose. Samples can be pre-treated by defatting (Sogami, M. and J. F. Foster (1968). Biochemistry 7(6): 2172-82, incorporated herein by reference) followed by thiol blocking (Sogami, M., H. A. Petersen, et al. (1969). Biochemistry 8(1): 49-58, incorporated herein by reference) and subsequent gel permeation chromatography. The binding curves generated for thio-albumins and HSA with octanoate, for example, may subsequently be compared, and functional similarity established.
- The albumin mutein (thio-albumin) of the invention can be covalently linked to one or more conjugation partners such as bioactive compounds by methods known in the art (for example those provided by Pierce, Thermo Fisher Scientific, Rockford, IL, USA; http://www.piercenet.com/files/1601361Crosslink.pdf). These include, but are not limited to incorporating or engineering a thiol reactive group into or onto the conjugation partner, for example by incorporating or engineering another free thiol present on the conjugation partner; or by incorporating or engineering a pyridyl disulphide group on the conjugation partner; or by incorporating or engineering an iodoacetyl group on the bioactive compound or or by incorporating or engineering a maleimide group on the conjugation partner. For example, N-ethylmaleimide (NEM, Pierce), 2-amino-2′-aminoethanethiolsulfonate (Pierce), N-beta-maleimidoprpionic acid (BMPA Pierce), methyl methane thiosulfonate (MMTS, Pierce), fluorescein-5-maleimide (Pierce), 5-iodoacetamido-fluorescein (5-IAF, Pierce) or N-[6-7-amino-4-methylcoumarin-3-acetamido) hexyl]-3′[2′-pyridyldithio] propionamide (AMCA-HPDP, Pierce).
- If the conjugation partner contains at least one thiol group, then the conjugation partner may be cross-linked to the albumin mutein of the invention by methods known to the art such as, but not limited to, oxidation or by the use of cross-linking reagents such as, but not limited to, 1,4-Bis-maleimidibutane (BMB, Pierce); 1,4-Bis-maleimidyl-2,3-dihydroxybutane (BMDB, Pierce); Bis-maleimidohexane (BMH, Pierce), Bis-maleimidoethane (BMOE, Pierce); 1,8-Bis-Maleimidotriethyleneglycol (BM[PEO]3 Pierce); 1,11-Bis-Maleimidotetraethyleneglycol (BM[PEO]4 Pierce); 1,4-Di-[3′-(2′-pyridyldithio)-propionamido]butane (DPDPB, Pierce); dithuio-bis-maleimidoethane (DTME Pierce); 1,6-Hexane-bis-vinylsulfone (HBVS, Pierce) and Tris-[2-maleimimidoethyl]amine (TMEA, Pierce).
- If the conjugation partner does not contain a thiol reactive group then it may be modified to incorporate one or more such groups by either chemical modification or genetic engineering by methods know to the art (Chapman, A.P. (2002) Adv. Drug Deliv. Rev., 54 531-545: Humphreys, D. P. et al. Protein Engineering, Design & Selection vol. 20 no. 5 pp. 227-234, 2007). While these two references describe methodologies to cross-link PEG to an engineered free thiol within an antibody or antibody fragment, the techniques may be used to cross-link a conjugation partner to an engineered free thiol within the albumin mutein of the invention. Alternatively the Drug Affinity Complex (DAC™) technology developed by ConjuChem Inc. (Montreal, Quebec, Canada, H2X 3Y8) may be used, e.g. as described in WO200069902. There are three parts of each DAC™ construct: 1) the drug component (the portion responsible for biologic activity); 2) a linker attached to the drug component, and 3) a reactive chemistry group at the opposite end of the linker, usually a soft electrophile selective for thiols; a maleimide is the most useful embodiment. Other applicable conjugation methods are described in WO2007/071068 incorporated herein by reference.
- If the conjugation partner does not contain a thiol reactive group but does contain one or more amino groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-5-azido-2-nitrobenzoyloxysuccinimide (AMAS, Pierce), N-[beta-maleimidopropyloxy] succinimide ester (BMPS, Pierce), N-eta-maleimidocaproic acid (EMCA, Pierce), N-[eta-maleimidocaproyloxy]succinimide ester (EMCS, Pierce), N-[eta-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS, Pierce), N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS, Pierce), N-[gamma-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS, Pierce), N-kappa-maleimidoundecanoic acid (KMUA, Pierce), N-[kappa -maleimidoundecanoic acid]hydrazide (KMUH, Pierce), N-[kappa -maleimidoundecanoyloxy]sulfosuccinimide ester (sulfo-KMUS, Pierce), m-maleimidobenzoyl-N-hydroxysuccinimide (MBS, Pierce), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS, Pierce), N-succinimidyl S-acetylthio-acetate (SATA, Pierce), N-succinimidyl S-acetylthiopropionate (SATP, Pierce), succinimidyl 3-[bromoacetamido]propionate (SBAP, Pierce), N-succinimidyl iodoacetate (SIA, Pierce), N-succinimidyl[4-iodoacetyl]aminobenzoate (STAB, Pierce), sulfosuccinimidyl[4-iodoacetyl]aminobenzoate (sulfo-SIAB, Pierce), succinimidyl [4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC, Pierce), sulfosuccinimidyl [4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC, Pierce), succinimidyl-[4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate (LC-SMCC, Pierce), 4-succininimidyloxycarbonyl-methyl-alpha[2-pyridyldithio]toluene (SMPT, Pierce), sulfosuccinimidyl-6-[alpha-methyl-alpha□2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT, Pierce), succinimidyl 4-[p-maleimidophenyl]-butyrate (SMPB, Pierce), sulfosuccinimidyl 4-[p-maleimidophenyl]-butyrate (sulfo-SMPB, Pierce), succinimidyl-6-[(beta-maleimidopropionamido)hexanoate] (SMPH, Pierce), N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP, Pierce), succinimidyl [3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP, Pierce), sulfosuccinimidyl [3′-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP, Pierce) and N-succinimidyl-[4-vinylsulfonyl]benzoate (SVSB Pierce). It may be advantageous to block certain amine residue as described by Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384.
- If the conjugation partner does not contain a thiol reactive group but does contain one or more carbonyl (oxidised carbohydrate) groups then it can be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[eta-maleimidocaproic acid]hydrazide (EMCH, Pierce), 4-[N-maleimidomethyl]cyclohexane-1carboxylhydrazide.HCl.1/2 dioxane (M2C2H, Pierce), 3-maleimidophenyl boronic acid (MPBH, Pierce) and 3-[2-pyridyldithio]propionyl hydrazide (PDPH, Pierce).
- If the conjugation partner does not contain a thiol reactive group but does contain one or more hydroxyl groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[p-maleimidophenyl]isocyanate (PMPI, Pierce).
- The conjugation competence of polypeptides of the invention may be tested by fluorescent labelling and cellular uptake, as described by McGraw et al., (1987), The Journal of Cell Biology, 105, 207-214 and Presley et al., (1993), The Journal of Cell Biology, 122, 1231-1241. Other methods of testing conjugation competence include conjugating the albumin to another molecule such as HRP. Subsequently, the mass of the resultant conjugate and/or the activity of the conjugated compound may be assayed, for example by mass spectrometry or by enzyme assay.
- A host strain suitable for use in the present invention includes an hsp150-deficient version of DXY1, disclosed in S. M. Kerry-Williams et al. (1998) Yeast 14:161-169. WO 95/33833 teaches the skilled person how to prepare hsp150-deficient yeast. This host strain may be referred to as ‘Strain 1’.
- All documents cited are incorporated by reference in their entirety.
- The invention is described by way of example only with reference to the following examples:
- The HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example,
EP 0 073 646 andEP 0 286 424. Expression plasmids for albumin variants of this invention can be constructed in a similar way to pDB2244 described in WO 00/44772 or pDB2305 described in WO/2006/013859 for expression of human serum albumin from S. cerevisiae. Plasmid pDB2305 contains the HSA sequence codon-optimised for expression in S. cerevisiae. Alternative codon optimisation methods may be used for the particular host organism selected for thio-albumin production. Expression plasmids for albumin variants of this invention can also be constructed in a similar way to those described in WO 2005/061719 A1 for improved expression of human serum albumin from S. cerevisiae. - Thio-albumin muteins can be made following modification of plasmid pDB2244 (
FIG. 7 ) or pDB2305 by site directed mutagenesis. Overlapping mutagenic oligonucleotide sequences can be used to modify the codon of the selected residue(s) to any DNA sequence which encodes a cysteine residue (TGT or TGC) using the procedures indicated by a commercially available kit (such as Stratagene's Quikchange™ Kit). Alternatively, synthetic DNA fragments can be manufactured containing the desired modifications to the polynucleotide sequence. - Subcloning plasmids which may be used to create plasmid pDB2244 (
FIG. 7 ) are plasmid pDB2243 (FIG. 8 ) (described in WO 00/44772) and pSAC35 (described in EP 286424). Plasmids pDB2243 and pDB2244 contain the native HSA gene. A skilled person will appreciate that the expression cassette may or may not be codon optimised; methods for constructing expression plasmids containing HSA codon optimised for expression in S. cerevisiae are described in WO/2006/013859. The native nucleotide sequence encoding HSA is provided in SEQ ID No. 2. A HSA nucleotide sequence codon-optimised for expression in S. cerevisiae is provided as SEQ ID No. 3. - Plasmid pDB2243 (6.203 kb) was digested to completion using restriction endonucleases Notl to release the 2.992 kb human serum albumin expression cassette.
- Plasmid pSAC35 is derivative of pSAC3 by Chinery and Hinchliffe (1989) Curr. Genet. 16 , 21-25, and in EP 286424. Plasmid pSAC35 (11.037 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce 14.037 kb pDB2244 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene (
FIG. 7 ). A person skilled in the art will appreciate that the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention. - Alternatively plasmid pDB2690 may be used. The construction of plasmid pDB2690 is described in WO/2005061719 A1. Plasmid pDB2690 (13.018 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce a 16.039 kb plasmid pDB2713 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene (
FIG. 9 ). A person skilled in the art will appreciate that the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention. - As an alternative to site-directed mutagenesis expression plasmids for thio-albumin (i.e. conjugation competent albumin) variants of this invention could be made by subcloning synthesized DNA fragments into plasmid pDB2243 (
FIG. 8 ) prior to cloning into pSAC35 or pDB2690. A method for the construction of a thio-albumin subcloning plasmid containing one extra conjugation competent cysteine (relative to SEQ ID No. 1) is described, by way of example only, below - The albumin DNA sequence of pDB2243 includes two Hindi II restriction endonuclease sites.
- The synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected codon to a cysteine codon, or an existing cysteine codon is deleted or modified to a codon for another amino acid. Alternatively, the coding sequence for the mature thio-albumin may be extended at the 5′ or 3′ end(s) or insertions made within the polypeptide to add novel sequence(s) coding for cysteine or polypeptides containing one or more cysteine.
- Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected cysteine codon to an alternative codon to create an unpaired cysteine. Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified by substitution of two codons at a specified site to a cysteine codon (the amino acid chain length is reduced). Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence (e.g. SEQ ID No. 2 or SEQ ID No. 3 in relation to HSA) is modified by insertion of a cysteine codon at a specified site (the amino acid chain length is increased). Plasmid pDB2243 may be digested to completion with Hindlll restriction endonuclease and the fragment (approximately 4.383 kb) is recovered and dephosphorylated, the synthetic DNA containing the appropriate modification to the human serum albumin encoding sequence may then be cloned to produce the required thio-albumin subcloning plasmid. The thio-albumin subcloning plasmid may then be digested to produce an expression cassette, which may be cloned into a suitable expression plasmid in a similar manner to the construction of pDB2244, pDB2305 or pDB2713.
- Those skilled in the art will appreciate that expression cassette for thio-albumin variants with additional modifications to the albumin protein sequence could be produced using a similar method to that described for the construction of a thio-albumin subcloning plasmid containing one extra conjugation competent cysteine (relative to SEQ ID No. 1).
- A S. cerevisiae strain,
e.g. Strain 1, may be transformed to leucine prototrophy with pDB2244 (WO 00/44772), or pDB2305 (WO/2006/013859) for expression of human serum albumin or the appropriate thio-albumin expression plasmids. Yeast may be transformed using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-1,protocol 2; Ito et al, 1983, J. Bacteriol., 153, 16; Elble, 1992, Biotechniques, 13, 18). Transformants may be selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates. The composition of BMMD is described by Sleep et al., 2002, Yeast, 18, 403. Cryopreserved stocks may be prepared in 20% (w/v) trehalose from 10 mL BMMD shake flask cultures (24 hours, 30° C., 200 rpm). - Thio-albumin variants with single amino acid changes were selected from Tables 5A, 5B and 6A. These variants were identified as the preferred mutations according to the methods described above. Details of each variant are given in
FIG. 11 , which provides a Construct Reference (e.g. TA1 for rHA A2C), the name of the plasmid encoding each thio-albumin variant expression construct and flanking sequences required for in vivo recombination by gap-repair, and the number given to a cryopreserved yeast stock (the yeast stock number) producing each thio-albumin variant. Details of the mutant codons compared to SEQ ID No. 2 are also provided, as are the SEQ ID numbers for each thio-albumin variant (DNA and protein). - To modify the amino acids in non-human serum albumins, the equivalent positions to a particular position in HSA may be determined from an alignment including human serum albumin (SEQ ID No. 1) such as
FIGS. 2 and 3 . The skilled person is familiar with alignments and can readily determine whether or not an amino acid in a sequence is equivalent to an amino acid in another sequence. For example, the position of the amino acid in the nonhuman albumin is not necessarily the same relative to the N-terminal end of HSA. For example, fromFIG. 2 position 239 of HSA is an alanine residue, whereas the corresponding residue of the bovine sequence is serine-238. Similarly, valine-479 of HSA corresponds to leucine-478 of sheep albumin. The plasmid pDB3927 (FIG. 12 ) was constructed from plasmid pDB2244 (FIG. 7 , WO 0044772A, ‘FL’: fusion leader sequence). pDB2244 was digested with restriction enzymes Swal and Hpal (both produce blunt ends) and self-ligated to form pDB3927. To create plasmid pDB3964 (FIG. 13 ) restriction enzyme sites were modified in the albumin DNA sequence (SEQ ID No. 2) of pDB3927 without modifying the protein sequence, as outlined below. The resultant DNA sequence is sequence ID No. 4. - 1) Introduced Enzyme Sites: (Basepair Positions in Brackets Refer to Positions in SEQ ID No. 4) SEQ ID No. 4
-
Restriction site SEQ ID No. SEQ ID No. 4 Position a SacII: GAGTCAGCTGAAAA →(to) GAGTCCGCGGAAAA (bp 173-178) b NheI/BmtI: AAGGCTTCGTCTGC →(to) AAGGCTAGCTCTGC (bp 571-576) c XhoI: TCTGCTTGAATGTGC →(to) TCTGCTCGAGTGTGC (bp 751-756) d BamHI: GTGGGCAGCAAAT →(to) GTGGGATCCAAAT (bp 751-756) e SalI: GGAAGTCGATGAAA →(to) GGAAGTCGACGAAA (bp 1477- 1482) - The coding sequence of HSA in pDB3964 is provided as SEQ ID No. 4. DNA synthesis and cloning was used to generate pDB3964 from pDB3927 (DNA2.0 Inc, USA). Synthetic DNA fragments were designed to alter specific amino acid codons within the albumin gene of pDB3964, or with combinations of modifications (see Example 3 below). DNA fragments containing these modifications were synthesised (DNA 2.0 Inc, USA) and cloned into pDB3964 to produce plasmids containing the thio-albumin sequences (
FIG. 11 ). These synthetic genes and flanking regions were excised with restriction enzymes BstEII and BsrBI from the plasmids named inFIG. 11 for each of the thio-albumin variants and the controls pDB3927, pDB3964 and pDB2244, before purification of the resulting DNA fragments (PCR purification kit, Qiagen). The DNA fragments were used in the yeast transformation procedure described below to allow gap-repair in vivo with linearised pDB3936. - The plasmid pDB3853 (not shown) was constructed from base vector pDB2690 (Ref DB88/WO2005/061719A1) and the synthetic linker described below. The synthetic linker was constructed from two oligonucleotides (Sigma-Genosys) annealed in distilled water using a temperature gradient from 96° C. to room temperature (1 min per 1° C.). pDB2690 was digested using Kpnl and Notl, and purified by gel extraction (Qiagen), before ligation of the annealed linker:
-
‘KpnI (linker) BamHI NotI’ 5′- CGCTAGCCTCGAGGTTTAAACGCTAGCGAGCTCGGATCC -3′ 3′- CATGGCGATCGGAGCTCCAAATTTGCGATCGCTCGAGCCTAGGCCG G -5′ - Following the construction of pDB3853, the linker was excised using Pstl and Scal (3787bp fragment) before ligation into the gel extracted Pstl/Scal cut pSAC35 plasmid (WO 0044772A and WO2005/061719A1)), to form pDB3936 (
FIG. 14 ). - pDB3936 was linearised with restriction enzymes Acc651 and BamHI before purification of the 9721bp fragment following separation by agarose gel electrophoresis. For the yeast transformation procedure to allow gap-repair in vivo (described below) the concentrations of the linearised pDB3936 and each of the BsrBI-BstEII fragments encoding the thio-albumin coding sequences was calculated and 100 ng of each use for each yeast transformation reaction.
- Saccharomyces cerevisiae strain BXP10 was used as the expression host throughout (So-low, S. P., J. Sengbusch, et al. (2005). “Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae.” Biotechnol Prog 21(2): 617-20.), although alternative expression hosts are also be suitable.
- Cryopreserved stocks of S. cerevisiae BXP10 were prepared from 10 mL YEPPD (1% w/v yeast extract, 2% w/v plant peptone, 2% w/v dextrose)) shake flask cultures (grown for 24 hours, 30° C., 200 rpm) mixed with an equal volume of 40% w/v sterile trehalose solution and dispensed in 1mL aliquots for storage at −80° C. 10 mL BMMD, YEPPD and LB (1% w/v bacteriological tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl) shake flasks were inoculated with 100 μL cryopreserved yeast stock and incubated for four days at 30° C., 200 rpm as above before being observed microscopically to confirm they were axenic.
- Frozen competent S. cerevisiae BXP10 cells were prepared by inoculating 100 μL cryopreserved yeast stock into 10 mL YEPPD which were incubated for two days at 30° C., 200 rpm, before being used to inoculate 300 mL YEPPD to an OD600=0.3. The cells were incubated as above for approximately 4 hours or until a doubling of OD600 had been achieved. The cells were harvested by centrifugation (3000×g, 5 min, room temperature) before resuspension in 120 mL distilled water followed by a further centrifugation step. The pellet was resuspended in 3 mL TE/LiAc (10 mM Tris, 1 mM EDTA, pH7; 500 mM lithium acetate) and glycerol added to a final concentration of 15% (v/v), before storage in aliquots at −80° C.
- S. cerevisiae BXP10 cells were transformed to leucine prototrophy using a modified lithium acetate method (Elble, R. “A simple and efficient procedure for transformation of yeasts.” Biotechniques 13.1 (1992): 18-20. Ito, H., et al. “Transformation of intact yeast cells treated with alkali cations.” J.Bacteriol. 153.1 (1983): 163-68.). 50 μl of thawed competent cells were aliquoted into a 48-well microtitre plate (Nunc) before the addition of DNA fragments for gap-repair, as described above. The plate was mixed by swirling of the plate while flat on a benchtop. 300 μl of PEG/LiAc (40% w/
v PEG 3350, 100 mM lithium acetate) was added to each well and was mixed again. The plate was incubated at 30° C. with shaking at 200 rpm for 1 hour before transfer to static incubation at 42° C. for 30 min. After 1 min incubation on ice, the plate was centrifuged (2000×g, 5 min, room temperature) followed by removal of the supernatant and resuspension of the pellet in 200 μl 1M sorbitol. The full volume was inoculated onto BMMD agar plates with CSM-Leu nutritional supplement (MP Biomedicals, Bio 101) and incubated for 4 days at 30° C. - Single colony transformants were picked and patched onto fresh BMMD agar plates for short term storage. These patches were grown at 30° C. and cells then inoculated into 10 mL BMMD shake flask cultures and cryopreserved as described earlier. 10 μl of yeast stock was inoculated into a 48-well plate containing 0.5 mL BMMD per well. Growth of cultures in microtitre plates was achieved in a humidity chamber which was a sealed Perspex box containing wet paper towels to provide ˜100% humidity and evaporative loss below 0.25% over 5 days under growth conditions. The plates were incubated in the shaking humidity chamber (30° C., 200 rpm,) for 5 days at 30° C. The 48-well plate was centrifuged to pellet cells (2000×g, 10 min, room temperature) and the supernatant was harvested.
- The concentration of the thio-albumin variants in the culture supernatants was determined by Gel Permeation High Pressure Liquid Chromatography (GP-HPLC). Protein concentrations were determined using a LC2010 HPLC system (Shimadzu) equipped with UV detection under Shimadzu VP7.3 client server software control. Injections of 25 μL were made onto a 7.8 mm internal diameter×300 mm length TSK G3000SWXL column (Tosoh Bioscience), with a 6.0 mm internal diameter x 40 mm length TSK SW guard column (Tosoh Bioscience). Samples were chromatographed in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL.min−1, with a run time of 15 minutes. Samples were quantified by UV detection at 280 nm, by peak height, relative to a recombinant human albumin standard of known concentration (10 mg/mL).
- A non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of the thio-albumin variants with single mutations are compared against controls in
FIG. 15 . It is evident that all of the thio-albumin variants have been successfully secreted from S. cerevisiae BXP10. Preferred mutations have high expression titres and show a sharp Coomassie stained band equivalent to rHA controls by non-reducing SDS-PAGE analysis. -
FIG. 16 describes an additional selection of thio-albumin variants with two or more free-thiol groups. Mutations shown to be expressed in Example 2 above were combined to generate sequences designed to have multiple free-thiol groups available for conjugation. This selection includes thio-albumin variants designed to have up to five free-thiol groups, thio-albumin variants designed to have free-thiol groups from within one Selection Group or from more than one Selection Group, thio-albumin variants designed to have free-thiol groups with and without the naturally occurring free-thiol at C34 of HSA, thio-albumin variants designed to have free-thiol groups from a range of Proximity Groups, and thio-albumin variants designed to have free-thiol groups derived from insertions, extensions, additions and/or deletions. It represents a sub-set of thio-albumins with multiple conjugation competent cysteine residues. The details of these thio-albumin variants, the plasmids encoding them and the SEQ ID No. for their DNA and protein sequences are described inFIG. 16 , in a similar manner to those ofFIG. 11 for the thio-albumin variants with single modifications. Methods for plasmid construction and expression from S. cerevisiae BXP10 are similar to those described above. -
FIG. 17 shows a non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of these additional thio-albumin variants compared against an rHA control (pDB3927 coding sequence). Again, it is evident that all of the thio-albumin variants have been successfully secreted from S. cerevisiae BXP10. It therefore confirms that the selection criteria allow suitable thio-albumin variants to be generated and therefore indicates that there is no problem with undersirable mis-folding or aggregation. Again, preferred combinations of mutations have high expression titres and show a sharp Coomassie stained band equivalent to rHA controls by non-reducing SDS-PAGE analysis. - Five cryopreserved yeast stocks (9116, 9118, 9124, 9125 and 9130;
FIG. 11 ) each in 1 mL aliquots were inoculated into shake flasks containing 100 mL BMMS growth medium (yeast nitrogen base without amino acids and (NH4)2SO4, Difco 1.7 g/L; citric acid monohydrate 6.09 g/L; anhydrous Na2HPO4 20.16 g/L; (NH4)2SO4 5.0 g/L; pH6.5±0.2; sucrose added to 20 g/L). Cells were transferred from the shake flask to the fermenter (10 L working volume, Sartorius Biostat C 10-3 fermenter) when the concentration of cells in the shake flask has reached 0.8-1.2 g/L achieving a cell inocula concentration of ≧10 mg/L (greater than or equal to 10 mg/L) in the fermenter. - The thio-albumin variants proteins were produced by axenic culture of each of the five yeast strains in high cell density (HCD) fed-batch fermentation. The aim of the fermentation was to achieve maximum biomass and productivity by controlling feed rate addition so that formation of byproducts such as ethanol and acetate were avoided. Further details of the fermentation process are described in WO96/37515. The temperature and pH were controlled at 30° C. and pH5.5 respectively. Culture supernatant was harvested by centrifugation using a Sorvall RC 3C centrifuge (DuPont) and frozen for storage, before being thawed for subsequent purification. Final product concentrations were determined by GP HPLC using a LC2010 HPLC system (Shimadzu) equipped with UV detection under Shimadzu VP7.3 client server software control as described above.
FIG. 18 provides the yields of each thio-albumin variant (in g/L culture supernatant) and shows that high product titres of greater that 1 g/L culture supernatant were obtained in all cases. - A single step chromatography procedure was used to prepare material suitable for mass spectrometry. This purification step used a column (bed volume approximately 200 μL) packed with AlbuPure™ matrix (ProMetic BioSciences Ltd, Cambridge UK or Novozymes Biophama UK Ltd.). This was equilibrated with 50 mM sodium phosphate, pH5.3, and loaded with neat culture supernatants, at approximately pH5.5-6.5, to approximately 40 mg protein/mL matrix. The column was washed with approximately 3 column volumes each of 50 mM sodium phosphate, pH5.3, and 50 mM ammonium acetate, pH8.0, respectively. Bound protein was eluted using approximately 5 column volumes of 50 mM ammonium acetate, 10 mM octanoate, pH7.0. The flow rate for the load step was 137 μL/min, while the wash and elution steps were performed by means of centrifugal force, using a
Heraeus Multifuge 3 centrifuge at 300 rpm. Final concentrations were in the range 1.8-4.0 mg/mL and samples were approximately 2 mL volume. Free thiol determination was performed immediately after sample elution by following the procedure described below. - The number of free thiols on a protein can be determined spectrophotometrically using Ellman's reagent. Ellman's reagent (5′5′-dithio-bis(2-nitronenzoic acid) (DTNB)) is an aromatic disulphide which reacts with thiol groups to form a mixed disulphide of the protein and one mole of 5-thio-2-nitrobenzoic acid (TNB) (per mole of protein sulfhydryl group). This reaction also results in a yellow colour from free TNB being released in solution. Alternatively the number of free thiols on a protein can be determined using mass spectrometric analysis of protein sample treated with DTNB reagent. 5-thio-2-nitrobenzoic acid (TNB) has a molecular weight of 199 Da, thus an increase in mass of 197 Da (TNB minus H2 lost during disulphide bond formation with the free thiol group on the test protein) indicates the presence of one free thiol group on the protein sample.
- 700 μL of the test protein sample was added to 100 μl Buffer 2 (4 mg/mL DTNB and 500 mM Sodium Phosphate, pH 7.0) and 900 μL Buffer 1 (0.1 M Tris-HCl, 100 mM EDTA, pH8.0). The preparation was allowed to mix for 25 minutes at ambient temperature (21-25° C.) followed by filtration through a low molecular mass cut-off filter (Vivaspin 2-10000 MWCO Sartorius Stedim Germany). The filter was washed with two volumes of 0.1% Trifluoroacetic acid (TFA) and the sample was resuspended in 1 ml of 0.1% TFA. TNB labelled and unlabelled samples were prepared for mass spectrometric analysis by desalt-ing/concentrating using Solid Phase Extaction (SPE). SPE columns were prepared by first wetting with 1 mL of 70% Acetonitrile (ACN Fisher)/0.1% TFA and then equilibrating ready for loading with 0.1% TFA. 1 mL of sample was loaded on the equilibrated SPE columns allowing time for the protein to bind. The bound protein and SPE columns were then washed three times in 1 mL of 0.1% Formic acid (Merck). Finally the bound protein was eluted into pre-washed 1 mL microfuge tube with 0. 5mL 70% ACN/0.1%FA.
- For Time-of-
Flight mass spectrometry 30 μL of sample was introduced into a hybrid quadru-pole time-of flight mass spectrometer (QqOaTOF, Applied Biosystems, QSTAR-XL®), equipped with an lonSpray™ source in positive ion mode, using flow injection analysis (FIA). The only instrument parameter that is actively tuned is the Decoupling Potential (DP), typically set to 250 V. Typically 2 minutes of sample scans are averaged. For protein analysis the TOF analyser is calibrated against protonated molecular ions of equine myoglobin (Sigma) and resolution is typically >14,000. Instrument control and data acquisition and processing were performed using Analyst™ QS v1.1 software (Applied Biosystems). - The results of the above analysis of the purified thio-albumin samples are described below. On addition of DTNB all samples quickly turned yellow as expected due to the presence of numerous free thiols. When the samples were visually compared to an equivalent sample of rHA, containing a single free thiol, the colour change observed for the thio-albumin samples was significantly more intense, strongly indicating the presence of multiple free thiols on each thio-albumin molecule. Results are summarised in
FIG. 18 , with increasing colour intensity increasing denoted by increased number of “+”. - The thio-albumin variants produced at higher fermentation yields were preferred for analysis by the mass spectroscopy method described above. Therefore, the recombinant proteins rHA (A2C, L585C) (total of 3 free thiols), rHA (D129C, C360S, L585C) (total of 4 free thiols), and A2C rHA-Cys (total of 3 free thiols) were analysed by ESI TOF (electrospray ionsation time of flight) mass spectrometry pre- and post-DTNB treatment to determine the numbers of free thiols present on each molecule.
- When rHA (A2C, L585C) was analysed pre-DTNB treatment (
FIG. 19 ) the major deconvoluted peaks observed were at 66633 Da and 66807 Da which corresponds to 172 Da and 346 Da above the expected mass of 66461 Da. These modifications were likely to be due to species, of ˜172 Da present in the growth media cross linking to the free thiols in rHA (A2C, L585C). Post DTNB treatment mass spectrometric analysis (FIG. 20 ) resulted in a major a deconvoluted peak at 67428 Da which was 376 Da above the expected mass for the protein with 3 free thiols. This extra mass is most likely to be due to an extra TNB linked to a free thiol and a further 179 Da, this strongly suggests the presence of a 4 free thiols and a possible a further thiol blocked with a species of ˜179 Da. Hence, the rHA (A2C, L558C) thio-albumin variant is particularly surprising in that it provides more than the expected number of reactive groups available for conjugation. Also present is a series of peaks ˜396 Da apart which are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA (A2C, L558C) molecule. This adduct formation is known to occur in the presence of excess DTNB. - When rHA (D129C, C360S, L585C) (total of 4 free thiols) was analysed pre-DTNB treatment (
FIG. 21 ) the major deconvoluted peaks observed were at 66575 Da and 66747 Da which corresponds to 172 Da and 344 Da above the expected mass of 66403 Da. - These modifications were likely to be due to molecules of ˜172 Da present in the growth media cross linking to the free thiols in rHA (D129C, C360S, L585C). Post DTNB treatment mass spectrometric analysis (
FIG. 22 ) resulted in a major a deconvoluted peak at 67564 Da which was 373 Da above the expected mass for the protein with 4 free thiols. This extra mass is most likely to be due to an extra TNB linked to a free thiol and a further 176 Da, this strongly suggests the presence of a 5 free thiols and a possible a further thiol blocked with a species of ˜176 Da. Hence, the rHA (D129C, C360S, L585C) thio-albumin variant is particularly surprising in that it has provided more than the expected number of groups available for conjugation. Also present are a series of peaks ˜396 Da apart these are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA D129C, C360S, L585C. This adduct formation is known to occur in the presence of excess DTNB. - Finally when A2C rHA-Cys (total of 3 free thiols) was analysed pre-DTNB treatment (
FIG. 23 ) the major deconvoluted peaks observed were at 66747 Da and 66919 Da which corresponds to 172 Da and 344 Da above the expected mass of 66574 Da. However also present was a smaller peak corresponding to the expected unmodified mass at 66575 Da. This mass spectra indicates the blocking of some free thiols while a proportion of the molecule is present containing the expected 3 free thiols. Post DTNB treatment mass spectrometric analysis (FIG. 24 ) resulted in a major a deconvoluted peak at 67142 Da which was 23 Da below the expected mass of 67165 Da of the protein labelled with 3 TNB molecules, this was likely due to the presence of two TNB molecules and a 175 Da modification, suggesting the presence of 3 thiols, one of which was blocked by the unknown ˜175 Da species. However on closer inspection ofFIG. 24 the presence ofsecondary peaks 23 Da above each species can be seen. These secondary peaks correspond to small shoulders in the raw data (data not shown) which are likely to indicate the presence of the molecule modified with 3 TNB molecules, indicating 3 free thiols. The other major species present are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA D129C, C360S, L585C. This adduct formation is known to occur in the presence of excess DTNB. - In conclusion, a range of thio-albumin variants have been produced with three or more conjugation competent cysteine residues. The conjugation competent cysteines can be in regions of that may or may not have secondary structure, and/or may or may not be generated from natural disulphide bonds, and/or may or may not be additional cysteines residues (such as cysteine residues extending from the natural C-terminus of HSA).
- Samples of TA35 (i.e. A2C, A364, D562 in addition to naturally occurring C34) and TA33 (i.e. A2C, L585C in addition to naturally occurring C34) were incubated at room-temperature for 24 hours and both formed gels.
Claims (1)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/280,733 US20170081389A1 (en) | 2009-02-11 | 2016-09-29 | Albumin variants and conjugates |
US16/827,356 US11555061B2 (en) | 2009-02-11 | 2020-03-23 | Albumin variants and conjugates |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP09152625.1 | 2009-02-11 | ||
EP09152625 | 2009-02-11 | ||
EP09152686 | 2009-02-12 | ||
EP09152686.3 | 2009-02-12 | ||
US15455509P | 2009-02-23 | 2009-02-23 | |
PCT/EP2010/051751 WO2010092135A2 (en) | 2009-02-11 | 2010-02-11 | Albumin variants and conjugates |
US201113201123A | 2011-08-11 | 2011-08-11 | |
US15/280,733 US20170081389A1 (en) | 2009-02-11 | 2016-09-29 | Albumin variants and conjugates |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/201,123 Division US9493545B2 (en) | 2009-02-11 | 2010-02-11 | Albumin variants and conjugates |
PCT/EP2010/051751 Division WO2010092135A2 (en) | 2009-02-11 | 2010-02-11 | Albumin variants and conjugates |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/827,356 Continuation US11555061B2 (en) | 2009-02-11 | 2020-03-23 | Albumin variants and conjugates |
Publications (1)
Publication Number | Publication Date |
---|---|
US20170081389A1 true US20170081389A1 (en) | 2017-03-23 |
Family
ID=42027719
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/201,123 Active US9493545B2 (en) | 2009-02-11 | 2010-02-11 | Albumin variants and conjugates |
US15/280,733 Abandoned US20170081389A1 (en) | 2009-02-11 | 2016-09-29 | Albumin variants and conjugates |
US16/827,356 Active US11555061B2 (en) | 2009-02-11 | 2020-03-23 | Albumin variants and conjugates |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/201,123 Active US9493545B2 (en) | 2009-02-11 | 2010-02-11 | Albumin variants and conjugates |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/827,356 Active US11555061B2 (en) | 2009-02-11 | 2020-03-23 | Albumin variants and conjugates |
Country Status (9)
Country | Link |
---|---|
US (3) | US9493545B2 (en) |
EP (2) | EP2396347B1 (en) |
JP (2) | JP5936112B2 (en) |
KR (1) | KR101722961B1 (en) |
CN (2) | CN102317315A (en) |
DK (1) | DK2396347T3 (en) |
ES (1) | ES2630253T3 (en) |
SG (2) | SG172789A1 (en) |
WO (1) | WO2010092135A2 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10233228B2 (en) | 2010-04-09 | 2019-03-19 | Albumedix Ltd | Albumin derivatives and variants |
US10329340B2 (en) | 2012-03-16 | 2019-06-25 | Albumedix Ltd | Albumin variants |
US10501524B2 (en) | 2012-11-08 | 2019-12-10 | Albumedix Ltd | Albumin variants |
US10633428B2 (en) | 2015-08-20 | 2020-04-28 | Albumedix Ltd | Albumin variants and conjugates |
US10696732B2 (en) * | 2009-10-30 | 2020-06-30 | Albumedix, Ltd | Albumin variants |
US10711050B2 (en) | 2011-11-18 | 2020-07-14 | Albumedix Ltd | Variant serum albumin with improved half-life and other properties |
WO2022032175A1 (en) | 2020-08-06 | 2022-02-10 | Cidara Therapeutics, Inc. | Methods for the synthesis of protein-drug conjugates |
WO2022032188A1 (en) | 2020-08-06 | 2022-02-10 | Cidara Therapeutics, Inc. | Methods for the synthesis of protein-drug conjugates |
WO2022133281A1 (en) | 2020-12-17 | 2022-06-23 | Cidara Therapeutics, Inc. | Compositions and methods for the treatment of human immunodeficiency virus |
US11555061B2 (en) | 2009-02-11 | 2023-01-17 | Albumedix, Ltd | Albumin variants and conjugates |
Families Citing this family (41)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8697650B2 (en) | 2010-02-16 | 2014-04-15 | Medimmune, Llc | HSA-related compositions and methods of use |
WO2012006624A2 (en) | 2010-07-09 | 2012-01-12 | Biogen Idec Hemophilia Inc. | Factor ix polypeptides and methods of use thereof |
WO2012059486A1 (en) | 2010-11-01 | 2012-05-10 | Novozymes Biopharma Dk A/S | Albumin variants |
US9045564B2 (en) | 2011-02-15 | 2015-06-02 | Medimmune, Llc | HSA-related compositions and methods of use |
CA2828811C (en) | 2011-03-03 | 2021-09-21 | Zymeworks Inc. | Multivalent heteromultimer scaffold design and constructs |
CA2830660A1 (en) * | 2011-05-05 | 2012-11-08 | Novozymes Biopharma Dk A/S | Albumin variants |
SG10201606161UA (en) | 2011-07-05 | 2016-09-29 | Novozymes Biopharma Dk As | Albumin Formulation and Use |
US9353172B2 (en) | 2011-07-18 | 2016-05-31 | Arts Biologics A/S | Long acting biologically active luteinizing hormone (LH) compound |
US20150133383A1 (en) | 2012-05-11 | 2015-05-14 | Prorec Bio Ab | Method for diagnosis and treatment of prolactin associated disorders |
JP6498601B2 (en) | 2012-07-13 | 2019-04-10 | ザイムワークス,インコーポレイテッド | Multivalent heteromultimeric scaffold designs and constructs |
US20160033523A1 (en) | 2013-02-16 | 2016-02-04 | Novozymes Biopharma Dk A/S | Pharmacokinetic animal model |
WO2014144549A1 (en) | 2013-03-15 | 2014-09-18 | Biogen Idec Ma Inc. | Factor ix polypeptide formulations |
CN111875670A (en) * | 2013-06-12 | 2020-11-03 | 法瑞斯生物技术有限公司 | Peptides having antagonistic activity against native CXCR4 |
WO2015001049A1 (en) | 2013-07-04 | 2015-01-08 | Novartis Ag | O-mannosyltransferase deficient filamentous fungal cells and methods of use thereof |
PL3063275T3 (en) | 2013-10-31 | 2020-03-31 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
AU2015293949B2 (en) | 2014-07-21 | 2019-07-25 | Teknologian Tutkimuskeskus Vtt Oy | Production of glycoproteins with mammalian-like N-glycans in filamentous fungi |
US20160045606A1 (en) * | 2014-08-18 | 2016-02-18 | Northwestern University | Partially-denatured protein hydrogels |
JP2017530153A (en) * | 2014-10-02 | 2017-10-12 | ウェイク フォレスト ユニバーシティー ヘルス サイエンシズ | Amniotic powder and its use in wound healing and tissue engineering constructs |
KR101637010B1 (en) * | 2015-04-24 | 2016-07-07 | 광주과학기술원 | Site-Specifically Albumin Conjugated Urate Oxidase and The Method for site-specifically conjugating albumin to Protein |
EP3394243A1 (en) | 2015-12-22 | 2018-10-31 | Albumedix Ltd. | Improved protein expression strains |
BR112018067371A2 (en) | 2016-03-02 | 2019-01-15 | Novozymes As | cellobiohydrolase variant, enzyme composition, whole broth formulation or cell culture composition, polynucleotide, recombinant host cell, plant, plant part or transgenic plant cell, methods for producing and obtaining a cellobio variant. hydrolase, transgenic plant, and processes for degradation of a cellulosic material, for production of a fermentation product and for fermentation of a cellulosic material |
CN109072209A (en) | 2016-03-24 | 2018-12-21 | 诺维信公司 | Cellobiohydrolase variant and the polynucleotides for encoding it |
RU2740913C2 (en) | 2016-04-29 | 2021-01-21 | Дефенсин Терапьютикс Апс | Treatment of liver, bile duct and pancreatic disorders |
WO2018026742A1 (en) * | 2016-08-01 | 2018-02-08 | Askgene Pharma Inc. | Novel antibody-albumin-drug conjugates (aadc) and methods for using them |
KR102662031B1 (en) | 2016-10-04 | 2024-05-03 | 알부메딕스 리미티드 | Uses of recombinant yeast-derived serum albumin |
US20210333279A1 (en) | 2016-11-04 | 2021-10-28 | Aarhus Universitet | Identification and treatment of tumors characterized by an overexpression of the neonatal fc receptor |
CA3045079A1 (en) | 2016-12-13 | 2018-06-21 | Defensin Therapeutics Aps | Methods for treating inflammatory conditions of the lungs |
JP7282693B2 (en) | 2017-06-20 | 2023-05-29 | アルブミディクス リミティド | Improved protein expression strain |
EP3684811A2 (en) | 2017-08-17 | 2020-07-29 | Massachusetts Institute of Technology | Multiple specificity binders of cxc chemokines and uses thereof |
KR101964376B1 (en) | 2017-09-29 | 2019-04-01 | 주식회사 나이벡 | Pharmaceutical composition and Biomaterial Containing Fusion Peptide Comprising Parathyroid hormone(PTH) bonded Bone-selective Peptide |
KR20200095472A (en) | 2017-11-10 | 2020-08-10 | 디펜신 테라퓨틱스 에이피에스 | Mucosal defense and maturation of intestinal/lung function in preterm infants |
US20200291082A1 (en) | 2017-11-24 | 2020-09-17 | Defensin Therapeutics Aps | Prevention and treatment of graft-versus-host-disease with defensins |
EP3847246A1 (en) | 2018-09-06 | 2021-07-14 | Bavarian Nordic A/S | Storage improved poxvirus compositions |
US20230000774A1 (en) | 2019-12-04 | 2023-01-05 | Albumedix Limited | Methods and compositions produced thereby |
CN111072784B (en) * | 2019-12-30 | 2022-12-13 | 中山大学附属第五医院 | Macromolecular furin inhibitor and preparation method and application thereof |
CN111118018B (en) * | 2020-03-05 | 2021-06-01 | 泰州博莱得利生物科技有限公司 | Cat serum albumin recombinant protein and expression method thereof in pichia pastoris |
MX2022011071A (en) | 2020-03-12 | 2022-09-23 | Bavarian Nordic As | Compositions improving poxvirus stability. |
WO2021246557A1 (en) * | 2020-06-05 | 2021-12-09 | 주식회사 프로앱텍 | Urate oxidase-albumin conjugate having certain number of conjugated albumins, and method for producing same |
EP4282876A1 (en) * | 2020-09-25 | 2023-11-29 | Proabtech Inc. | Uricase-albumin conjugate, preparation method therefor, and use thereof |
WO2023079553A1 (en) * | 2021-11-03 | 2023-05-11 | Biond Biologics Ltd. | Intracellular delivery compositions |
EP4442251A1 (en) | 2023-04-05 | 2024-10-09 | Albumedix Ltd | Formulations and uses thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7141547B2 (en) * | 2001-12-21 | 2006-11-28 | Human Genome Sciences, Inc. | Albumin fusion proteins comprising GLP-1 polypeptides |
US20090029914A1 (en) * | 2006-06-07 | 2009-01-29 | Rosen Craig A | Albumin Fusion Proteins |
Family Cites Families (179)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5625041A (en) | 1990-09-12 | 1997-04-29 | Delta Biotechnology Limited | Purification of proteins |
GB9019919D0 (en) | 1990-09-12 | 1990-10-24 | Delta Biotechnology Ltd | Purification of proteins |
US2714586A (en) | 1951-06-25 | 1955-08-02 | Phillips Petroleum Co | Washing urea and thiourea containing adducts |
US4302386A (en) | 1978-08-25 | 1981-11-24 | The Ohio State University | Antigenic modification of polypeptides |
NZ199722A (en) | 1981-02-25 | 1985-12-13 | Genentech Inc | Dna transfer vector for expression of exogenous polypeptide in yeast;transformed yeast strain |
IL66614A (en) | 1981-08-28 | 1985-09-29 | Genentech Inc | Method of constructing a dna sequence encoding a polypeptide,microbial production of human serum albumin,and pharmaceutical compositions comprising it |
US4741900A (en) | 1982-11-16 | 1988-05-03 | Cytogen Corporation | Antibody-metal ion complexes |
US4757006A (en) | 1983-10-28 | 1988-07-12 | Genetics Institute, Inc. | Human factor VIII:C gene and recombinant methods for production |
JPS60169498A (en) * | 1984-02-10 | 1985-09-02 | Kyowa Hakko Kogyo Co Ltd | Adult t cell leukemic viral antigen peptide derivative |
US4885249A (en) | 1984-12-05 | 1989-12-05 | Allelix, Inc. | Aspergillus niger transformation system |
WO1986006101A1 (en) | 1985-04-12 | 1986-10-23 | Genetics Institute, Inc. | Novel procoagulant proteins |
DE3650783T2 (en) | 1985-04-15 | 2004-07-15 | Dsm Ip Assets B.V. | Use of the Apergillus glucoamylase promoter |
GR860984B (en) | 1985-04-17 | 1986-08-18 | Zymogenetics Inc | Expression of factor vii and ix activities in mammalian cells |
US5364770A (en) | 1985-08-29 | 1994-11-15 | Genencor International Inc. | Heterologous polypeptides expressed in aspergillus |
DK122686D0 (en) | 1986-03-17 | 1986-03-17 | Novo Industri As | PREPARATION OF PROTEINS |
GB8615701D0 (en) | 1986-06-27 | 1986-08-06 | Delta Biotechnology Ltd | Stable gene integration vector |
GB8620926D0 (en) | 1986-08-29 | 1986-10-08 | Delta Biotechnology Ltd | Yeast promoter |
DE3888381T2 (en) | 1987-04-09 | 1994-07-28 | Delta Biotechnology Ltd | Yeast vector. |
ES2076939T3 (en) | 1987-08-28 | 1995-11-16 | Novo Nordisk As | RECOMBINANT LUMPY OF HUMICOLA AND PROCEDURE FOR THE PRODUCTION OF RECOMBINANT LIPAS OF HUMICOLA. |
GB8725529D0 (en) | 1987-10-30 | 1987-12-02 | Delta Biotechnology Ltd | Polypeptides |
IL88326A (en) | 1987-11-18 | 1993-03-15 | Gist Brocades Nv | Purification of serum albumin |
JPH01215289A (en) | 1988-02-22 | 1989-08-29 | Toa Nenryo Kogyo Kk | Production of normal human serum albumin a through gene recombination |
US5075222A (en) | 1988-05-27 | 1991-12-24 | Synergen, Inc. | Interleukin-1 inhibitors |
ATE135045T1 (en) | 1988-07-23 | 1996-03-15 | Delta Biotechnology Ltd | SECRETORY LEADER SEQUENCES |
FR2649991B2 (en) | 1988-08-05 | 1994-03-04 | Rhone Poulenc Sante | USE OF STABLE DERIVATIVES OF PLASMID PKD1 FOR THE EXPRESSION AND SECRETION OF HETEROLOGOUS PROTEINS IN YEASTS OF THE GENUS KLUYVEROMYCES |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5759802A (en) | 1988-10-26 | 1998-06-02 | Tonen Corporation | Production of human serum alubumin A |
FR2646438B1 (en) | 1989-03-20 | 2007-11-02 | Pasteur Institut | A METHOD FOR SPECIFIC REPLACEMENT OF A COPY OF A GENE PRESENT IN THE RECEIVER GENOME BY INTEGRATION OF A GENE DIFFERENT FROM THAT OR INTEGRATION |
US5766883A (en) | 1989-04-29 | 1998-06-16 | Delta Biotechnology Limited | Polypeptides |
GB8909916D0 (en) | 1989-04-29 | 1989-06-14 | Delta Biotechnology Ltd | Polypeptides |
US5571697A (en) | 1989-05-05 | 1996-11-05 | Baylor College Of Medicine Texas Medical Center | Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus |
FR2650598B1 (en) | 1989-08-03 | 1994-06-03 | Rhone Poulenc Sante | DERIVATIVES OF ALBUMIN WITH THERAPEUTIC FUNCTION |
GB8927480D0 (en) | 1989-12-05 | 1990-02-07 | Delta Biotechnology Ltd | Mutant fungal strain detection and new promoter |
US5073627A (en) | 1989-08-22 | 1991-12-17 | Immunex Corporation | Fusion proteins comprising GM-CSF and IL-3 |
GB8923521D0 (en) | 1989-10-18 | 1989-12-06 | Delta Biotechnology Ltd | Yeast promoter |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
GB8927722D0 (en) | 1989-12-07 | 1990-02-07 | British Bio Technology | Proteins and nucleic acids |
DE4000939A1 (en) | 1990-01-15 | 1991-07-18 | Brem Gottfried Prof Dr Dr | METHOD FOR OBTAINING ANTIBODIES |
JP3230091B2 (en) | 1990-06-25 | 2001-11-19 | ウェルファイド株式会社 | Method for suppressing coloration of human serum albumin |
IL99552A0 (en) | 1990-09-28 | 1992-08-18 | Ixsys Inc | Compositions containing procaryotic cells,a kit for the preparation of vectors useful for the coexpression of two or more dna sequences and methods for the use thereof |
US5698426A (en) | 1990-09-28 | 1997-12-16 | Ixsys, Incorporated | Surface expression libraries of heteromeric receptors |
CA2058820C (en) | 1991-04-25 | 2003-07-15 | Kotikanyad Sreekrishna | Expression cassettes and vectors for the secretion of human serum albumin in pichia pastoris cells |
US5264586A (en) | 1991-07-17 | 1993-11-23 | The Scripps Research Institute | Analogs of calicheamicin gamma1I, method of making and using the same |
FR2686899B1 (en) | 1992-01-31 | 1995-09-01 | Rhone Poulenc Rorer Sa | NOVEL BIOLOGICALLY ACTIVE POLYPEPTIDES, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM. |
US5663060A (en) | 1992-04-07 | 1997-09-02 | Emory University | Hybrid human/animal factor VIII |
ZA932522B (en) | 1992-04-10 | 1993-12-20 | Res Dev Foundation | Immunotoxins directed against c-erbB-2(HER/neu) related surface antigens |
DE4226971C2 (en) | 1992-08-14 | 1997-01-16 | Widmar Prof Dr Tanner | Modified fungal cells and processes for the production of recombinant products |
US5728553A (en) | 1992-09-23 | 1998-03-17 | Delta Biotechnology Limited | High purity albumin and method of producing |
PL174721B1 (en) | 1992-11-13 | 1998-09-30 | Idec Pharma Corp | Monoclonal antibody anty-cd2 |
WO1995003402A1 (en) | 1993-07-22 | 1995-02-02 | Merck & Co., Inc. | EXPRESSION OF HUMAN INTERLEUKIN-1β IN A TRANSGENIC ANIMAL |
DE4343591A1 (en) | 1993-12-21 | 1995-06-22 | Evotec Biosystems Gmbh | Process for the evolutionary design and synthesis of functional polymers based on shape elements and shape codes |
US6811773B1 (en) | 1993-12-22 | 2004-11-02 | Human Genome Sciences, Inc. | Human monocyte colony inhibitory factor (M-CIF) polypeptides |
US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
GB9404270D0 (en) | 1994-03-05 | 1994-04-20 | Delta Biotechnology Ltd | Yeast strains and modified albumins |
US5773001A (en) | 1994-06-03 | 1998-06-30 | American Cyanamid Company | Conjugates of methyltrithio antitumor agents and intermediates for their synthesis |
GB9411356D0 (en) | 1994-06-07 | 1994-07-27 | Delta Biotechnology Ltd | Yeast strains |
US7597886B2 (en) | 1994-11-07 | 2009-10-06 | Human Genome Sciences, Inc. | Tumor necrosis factor-gamma |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US5955349A (en) | 1996-08-26 | 1999-09-21 | Zymogenetics, Inc. | Compositions and methods for producing heterologous polypeptides in Pichia methanolica |
US5716808A (en) | 1995-11-09 | 1998-02-10 | Zymogenetics, Inc. | Genetic engineering of pichia methanolica |
GB9526733D0 (en) | 1995-12-30 | 1996-02-28 | Delta Biotechnology Ltd | Fusion proteins |
US6509313B1 (en) | 1996-02-28 | 2003-01-21 | Cornell Research Foundation, Inc. | Stimulation of immune response with low doses of cytokines |
US5854039A (en) | 1996-07-17 | 1998-12-29 | Zymogenetics, Inc. | Transformation of pichia methanolica |
US5736383A (en) | 1996-08-26 | 1998-04-07 | Zymogenetics, Inc. | Preparation of Pichia methanolica auxotrophic mutants |
US6274305B1 (en) | 1996-12-19 | 2001-08-14 | Tufts University | Inhibiting proliferation of cancer cells |
US6605699B1 (en) | 1997-01-21 | 2003-08-12 | Human Genome Sciences, Inc. | Galectin-11 polypeptides |
US7053190B2 (en) | 1997-03-07 | 2006-05-30 | Human Genome Sciences, Inc. | Secreted protein HRGDF73 |
US7196164B2 (en) | 1997-07-08 | 2007-03-27 | Human Genome Sciences, Inc. | Secreted protein HHTLF25 |
US6506569B1 (en) | 1997-05-30 | 2003-01-14 | Human Genome Sciences, Inc. | Antibodies to human tumor necrosis factor receptor TR10 |
US5948609A (en) | 1997-12-03 | 1999-09-07 | Carter; Daniel C. | Oxygen-transporting albumin-based blood replacement composition and blood volume expander |
AU754237B2 (en) | 1998-08-06 | 2002-11-07 | Syntron Bioresearch, Inc. | Uric acid assay device with stabilized uricase reagent composition |
AU3128000A (en) | 1998-12-23 | 2000-07-31 | Human Genome Sciences, Inc. | Peptidoglycan recognition proteins |
GB9902000D0 (en) | 1999-01-30 | 1999-03-17 | Delta Biotechnology Ltd | Process |
DK2278016T3 (en) | 1999-03-22 | 2012-11-26 | Novozymes Inc | Fusarium Venenatum promoter sequences and their use |
WO2000069902A1 (en) | 1999-05-17 | 2000-11-23 | Conjuchem, Inc. | Long lasting fusion peptide inhibitors or viral infection |
CA2371912C (en) | 1999-05-21 | 2010-02-16 | American Bioscience, Inc. | Protein stabilized pharmacologically active agents, methods for the preparation thereof and methods for the use thereof |
AU5471501A (en) | 2000-03-22 | 2001-10-03 | Octagene Gmbh | Production of recombinant blood clotting factors in human cell lines |
AU2001266557A1 (en) | 2000-04-12 | 2001-10-23 | Human Genome Sciences, Inc. | Albumin fusion proteins |
WO2002016411A2 (en) | 2000-08-18 | 2002-02-28 | Human Genome Sciences, Inc. | Binding polypeptides and methods based thereon |
ATE510850T1 (en) | 2000-09-15 | 2011-06-15 | Coley Pharm Gmbh | METHOD FOR HIGH-THROUGHPUT SCREENING OF CPG-BASED IMMUNAGONISTS AND ANTAGONISTS |
AU2002245204A1 (en) | 2000-10-25 | 2002-07-24 | Genzyme Corporation | Methods for treating blood coagulation disorders |
WO2002043658A2 (en) | 2000-11-06 | 2002-06-06 | The Jackson Laboratory | Fcrn-based therapeutics for the treatment of auto-immune disorders |
AU2002226897B2 (en) | 2000-12-07 | 2007-10-25 | Eli Lilly And Company | GLP-1 fusion proteins |
US7175988B2 (en) | 2001-02-09 | 2007-02-13 | Human Genome Sciences, Inc. | Human G-protein Chemokine Receptor (CCR5) HDGNR10 |
US7507413B2 (en) | 2001-04-12 | 2009-03-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
DE60236646D1 (en) | 2001-04-13 | 2010-07-22 | Human Genome Sciences Inc | Anti-VEGF-2 antibodies |
AUPR446701A0 (en) | 2001-04-18 | 2001-05-17 | Gene Stream Pty Ltd | Transgenic mammals for pharmacological and toxicological studies |
US6949691B2 (en) | 2001-06-15 | 2005-09-27 | New Century Pharmaceuticals Inc. | Human albumin animal models for drug evaluation, toxicology and immunogenicity studies |
ATE375388T1 (en) | 2001-07-27 | 2007-10-15 | Us Gov Health & Human Serv | SYSTEMS FOR SITE-DIRECTED IN VIVO MUTAGENesis USING OLIGONUCLEOTIDES |
CN1405182A (en) | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
EP1463752A4 (en) | 2001-12-21 | 2005-07-13 | Human Genome Sciences Inc | Albumin fusion proteins |
US20080167238A1 (en) | 2001-12-21 | 2008-07-10 | Human Genome Sciences, Inc. | Albumin Fusion Proteins |
CA2475539A1 (en) | 2002-02-07 | 2003-08-14 | Delta Biotechnology Limited | Hiv inhibiting proteins |
US20080108560A1 (en) | 2002-03-05 | 2008-05-08 | Eli Lilly And Company | Heterologous G-Csf Fusion Proteins |
WO2003102136A2 (en) | 2002-05-30 | 2003-12-11 | Human Genome Sciences, Inc. | Antibodies that specifically bind to neurokinin b |
CN1241946C (en) | 2002-07-01 | 2006-02-15 | 美国福源集团 | Human serum albumins recombined merge protein having hyperplasia stimulation function to multiple cells |
GB0217347D0 (en) | 2002-07-26 | 2002-09-04 | Univ Edinburgh | Novel albumins |
AU2003257032A1 (en) | 2002-08-02 | 2004-02-23 | Human Genome Sciences, Inc. | Antibodies against c3a receptor |
WO2004058044A2 (en) | 2002-11-19 | 2004-07-15 | Drg International, Inc. | Diagnostic method for diseases by screening for hepcidin in human or animal tissues, blood or body fluids and therapeutic uses therefor |
CA2513213C (en) | 2003-01-22 | 2013-07-30 | Human Genome Sciences, Inc. | Albumin fusion proteins |
CA2516409A1 (en) | 2003-02-17 | 2004-08-26 | Upperton Limited | Conjugates for medical imaging comprising carrier, targeting moiety and a contrast agent |
GB0305989D0 (en) | 2003-03-15 | 2003-04-23 | Delta Biotechnology Ltd | Agent |
US20070041987A1 (en) | 2003-03-19 | 2007-02-22 | Daniel Carter | Fragments or polymers of albumin with tunable vascular residence time for use in therapeutic delivery and vaccine development |
CA2522680A1 (en) | 2003-04-15 | 2004-10-28 | Xenon Pharmaceuticals Inc. | Juvenile hemochromatosis gene (hfe2a), expression products and uses thereof |
US20050079546A1 (en) | 2003-05-01 | 2005-04-14 | Dasa Lipovsek | Serum albumin scaffold-based proteins and uses thereof |
WO2005007121A2 (en) | 2003-07-18 | 2005-01-27 | Massachusetts Institute Of Technology | Mutant interleukin-2(il-2) polypeptides |
US20080308744A1 (en) | 2003-11-18 | 2008-12-18 | Beth Israel Deaconess Medical Center | Serum Albumin Conjugated to Fluorescent Substances for Imaging |
GB0329722D0 (en) | 2003-12-23 | 2004-01-28 | Delta Biotechnology Ltd | Modified plasmid and use thereof |
GB0329681D0 (en) | 2003-12-23 | 2004-01-28 | Delta Biotechnology Ltd | Gene expression technique |
CA2486245C (en) | 2003-12-26 | 2013-01-08 | Nipro Corporation | Albumin having enhanced antimicrobial activity |
JP4649954B2 (en) * | 2003-12-26 | 2011-03-16 | ニプロ株式会社 | Albumin with enhanced antibacterial activity |
KR100671005B1 (en) | 2004-01-15 | 2007-01-18 | 고려대학교 산학협력단 | Biomarker proteins for diagnosing the exposure to PAH |
HUE027902T2 (en) | 2004-02-09 | 2016-11-28 | Human Genome Sciences Inc Corp Service Company | Albumin fusion proteins |
RU2369404C2 (en) | 2004-02-09 | 2009-10-10 | Хьюман Дженом Сайенсиз, Инк. | Fused proteins of albumine |
JP4492156B2 (en) | 2004-03-03 | 2010-06-30 | ニプロ株式会社 | Protein containing serum albumin domain |
US20060018859A1 (en) | 2004-07-16 | 2006-01-26 | Carter Daniel C | Modified human serum albumin with reduced or eliminated affinity to chemical or biological contaminants at Cys 34 |
WO2006013859A1 (en) | 2004-08-06 | 2006-02-09 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Yeast promoter |
US20060051859A1 (en) | 2004-09-09 | 2006-03-09 | Yan Fu | Long acting human interferon analogs |
AU2005286607B2 (en) * | 2004-09-23 | 2011-01-27 | Genentech, Inc. | Cysteine engineered antibodies and conjugates |
US20090280534A1 (en) | 2004-12-22 | 2009-11-12 | Novozymes A/S | Recombinant Production of Serum Albumin |
DK2330200T3 (en) | 2004-12-23 | 2017-07-24 | Albumedix As | GENE EXPRESSION TECHNIQUE |
WO2006073195A1 (en) | 2005-01-07 | 2006-07-13 | Biomarker Science Co., Ltd | Method for predicting or diagnosing diabetes and kit for predicting or diagnosing diabetes |
US20060178301A1 (en) | 2005-02-04 | 2006-08-10 | Mathias Jurs | Albumin-fused ciliary neurotrophic factor |
WO2006118772A2 (en) | 2005-04-29 | 2006-11-09 | The Jackson Laboratory | Fcrn antibodies and uses thereof |
GB0512707D0 (en) | 2005-06-22 | 2005-07-27 | Delta Biotechnology Ltd | Gene expression technique |
JP2009504157A (en) | 2005-08-12 | 2009-02-05 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | Albumin fusion protein |
CA2562249A1 (en) | 2005-10-20 | 2007-04-20 | University Of Ottawa Heart Institute | Anf analogue |
US20070269863A1 (en) | 2005-12-22 | 2007-11-22 | Bridon Dominique P | Process for the production of preformed conjugates of albumin and a therapeutic agent |
EP1816201A1 (en) | 2006-02-06 | 2007-08-08 | CSL Behring GmbH | Modified coagulation factor VIIa with extended half-life |
WO2007112940A2 (en) | 2006-03-31 | 2007-10-11 | Ablynx N.V. | Albumin-derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic compounds and entities, and constructs comprising the same |
ES2910207T3 (en) | 2006-06-14 | 2022-05-11 | Csl Behring Gmbh | Proteolytically Cleavable Fusion Proteins Comprising a Blood Coagulation Factor |
JP5670634B2 (en) | 2006-07-13 | 2015-02-18 | アッパートン リミティド | Method for producing particles of proteinaceous substance |
JP4983148B2 (en) | 2006-08-18 | 2012-07-25 | ニプロ株式会社 | Glycan-containing albumin, method for producing the same, and use thereof |
KR20090060294A (en) | 2006-09-08 | 2009-06-11 | 암브룩스, 인코포레이티드 | Modified human plasma polypeptide or fc scaffolds and their uses |
US20100129846A1 (en) | 2006-12-07 | 2010-05-27 | Power3 Medical Products, Inc. | Isoform specificities of blood serum proteins and their use as differentially expressed protein biomarkers for diagnosis of breast cancer |
CA2695830A1 (en) | 2007-08-08 | 2009-02-12 | Novozymes Biopharma Dk A/S | Transferrin variants and conjugates |
WO2009061853A2 (en) | 2007-11-05 | 2009-05-14 | Massachusetts Institute Of Technology | Mutant interleukin-2 (il-2) polypeptides |
CA2611540C (en) | 2007-11-09 | 2017-05-30 | Nipro Corporation | Sugar chain-containing albumin as a drug carrier to the liver |
KR101759457B1 (en) | 2007-12-21 | 2017-07-31 | 메디뮨 리미티드 | BINDING MEMBERS FOR INTERLEUKIN-4 RECEPTOR ALPHA (IL-4Rα)-173 |
KR20110008086A (en) | 2008-04-11 | 2011-01-25 | 메리맥 파마슈티컬즈, 인크. | Human serum albumin linkers and conjugates thereof |
JP2011523353A (en) | 2008-04-28 | 2011-08-11 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Overcharged protein for cell penetration |
KR20110052642A (en) | 2008-07-18 | 2011-05-18 | 오라제닉스, 인코포레이티드 | Compositions for the detection and treatment of colorectal cancer |
KR20110112301A (en) | 2008-11-18 | 2011-10-12 | 메리맥 파마슈티컬즈, 인크. | Human serum albumin linkers and conjugates thereof |
EP2373331A4 (en) | 2008-12-05 | 2015-11-18 | Abraxis Bioscience Llc | Albumin binding peptide-mediated disease targeting |
CN102307905B (en) | 2008-12-10 | 2015-11-25 | 斯克利普斯研究院 | Chemical reactivity alpha-non-natural amino acid is utilized to produce carrier-peptide conjugate |
US9493545B2 (en) | 2009-02-11 | 2016-11-15 | Albumedix A/S | Albumin variants and conjugates |
AU2010234459A1 (en) | 2009-04-08 | 2011-11-03 | The Regents Of The University Of California | Human protein scaffold with controlled serum pharmacokinetics |
JP2012525146A (en) | 2009-04-28 | 2012-10-22 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Overcharged protein for cell penetration |
WO2010138814A2 (en) | 2009-05-29 | 2010-12-02 | The Brigham And Women's Hospital, Inc. | Disrupting fcrn-albumin interactions |
EP2437767B1 (en) | 2009-06-01 | 2015-07-08 | MedImmune, LLC | Molecules with extended half-lives and uses thereof |
KR101286721B1 (en) | 2009-06-05 | 2013-07-16 | 한국과학기술연구원 | Recombinant albumins fused with poly-cysteine peptide and the methods for preparing the same |
EP2456454A4 (en) | 2009-07-20 | 2013-03-20 | Univ Nat Cheng Kung | Polypeptides selective for av 3 integrin conjugated with a variant of human serum albumin (hsa) and pharmaceutical uses thereof |
EP2456787A4 (en) | 2009-07-24 | 2013-01-30 | Univ Leland Stanford Junior | Cytokine compositions and methods of use thereof |
CA2770617C (en) | 2009-08-10 | 2018-02-20 | Mark Smith | Reversible covalent linkage of functional molecules |
US20110172398A1 (en) | 2009-10-02 | 2011-07-14 | Boehringer Ingelheim International Gmbh | Bispecific binding molecules for anti-angiogenesis therapy |
CA2777222A1 (en) | 2009-10-10 | 2011-04-14 | Eleven Biotherapeutics, Inc. | Il-17 family cytokine compositions and uses |
CA2776241A1 (en) | 2009-10-30 | 2011-05-05 | Novozymes Biopharma Dk A/S | Albumin variants |
EP2515926A4 (en) | 2009-12-23 | 2013-10-09 | Univ Nat Cheng Kung | Compositions and methods for the treatment of angiogenesis-related eye diseases |
CN101875693B (en) | 2010-01-22 | 2012-07-18 | 成都正能生物技术有限责任公司 | Albumin variant having anti-angiogenesis activity and preparation method thereof |
US8697650B2 (en) | 2010-02-16 | 2014-04-15 | Medimmune, Llc | HSA-related compositions and methods of use |
WO2011124718A1 (en) | 2010-04-09 | 2011-10-13 | Novozymes A/S | Albumin derivatives and variants |
CA2800173C (en) | 2010-05-21 | 2019-05-14 | Ulrik Nielsen | Bi-specific fusion proteins |
WO2011161127A1 (en) | 2010-06-21 | 2011-12-29 | Medimmune, Llc | Protease variants of human neprilysin |
JP2013538566A (en) | 2010-08-13 | 2013-10-17 | グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッド | Improved antiserum albumin binding variants |
WO2012059486A1 (en) | 2010-11-01 | 2012-05-10 | Novozymes Biopharma Dk A/S | Albumin variants |
US9045564B2 (en) | 2011-02-15 | 2015-06-02 | Medimmune, Llc | HSA-related compositions and methods of use |
EP2675471A4 (en) | 2011-02-15 | 2015-01-28 | Medimmune Llc | Hsa-related compositions and methods of use |
CA2830660A1 (en) | 2011-05-05 | 2012-11-08 | Novozymes Biopharma Dk A/S | Albumin variants |
US9353172B2 (en) | 2011-07-18 | 2016-05-31 | Arts Biologics A/S | Long acting biologically active luteinizing hormone (LH) compound |
US20140315817A1 (en) | 2011-11-18 | 2014-10-23 | Eleven Biotherapeutics, Inc. | Variant serum albumin with improved half-life and other properties |
BR112014018679A2 (en) | 2012-03-16 | 2017-07-04 | Novozymes Biopharma Dk As | albumin variants |
WO2014005596A1 (en) | 2012-07-03 | 2014-01-09 | Aarhus Universitet | Modified payload molecules and their interactions and uses |
CN105452290A (en) | 2012-11-08 | 2016-03-30 | 诺维信生物制药丹麦公司 | Albumin variants |
US20160033523A1 (en) | 2013-02-16 | 2016-02-04 | Novozymes Biopharma Dk A/S | Pharmacokinetic animal model |
US20160052993A1 (en) | 2013-05-03 | 2016-02-25 | Eleven Biotherapeutics, Inc. | Albumin variants binding to fcrn |
US20160222087A1 (en) | 2013-09-13 | 2016-08-04 | Novozymes Biopharma Dk A/S | Albumin variants |
CA2928700C (en) | 2013-11-01 | 2019-01-15 | University Of Oslo | Albumin variants and uses thereof |
JP7007261B2 (en) | 2015-08-20 | 2022-01-24 | アルブミディクス リミティド | Albumin variants and conjugates |
-
2010
- 2010-02-11 US US13/201,123 patent/US9493545B2/en active Active
- 2010-02-11 CN CN2010800074274A patent/CN102317315A/en active Pending
- 2010-02-11 ES ES10703295.5T patent/ES2630253T3/en active Active
- 2010-02-11 CN CN201710127505.8A patent/CN106986933A/en active Pending
- 2010-02-11 EP EP10703295.5A patent/EP2396347B1/en active Active
- 2010-02-11 SG SG2011046935A patent/SG172789A1/en unknown
- 2010-02-11 KR KR1020117019613A patent/KR101722961B1/en active IP Right Grant
- 2010-02-11 DK DK10703295.5T patent/DK2396347T3/en active
- 2010-02-11 EP EP17156183.0A patent/EP3243835B1/en active Active
- 2010-02-11 JP JP2011549569A patent/JP5936112B2/en active Active
- 2010-02-11 WO PCT/EP2010/051751 patent/WO2010092135A2/en active Application Filing
- 2010-02-11 SG SG2014012918A patent/SG2014012918A/en unknown
-
2016
- 2016-04-26 JP JP2016088309A patent/JP6279005B2/en active Active
- 2016-09-29 US US15/280,733 patent/US20170081389A1/en not_active Abandoned
-
2020
- 2020-03-23 US US16/827,356 patent/US11555061B2/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7141547B2 (en) * | 2001-12-21 | 2006-11-28 | Human Genome Sciences, Inc. | Albumin fusion proteins comprising GLP-1 polypeptides |
US20090029914A1 (en) * | 2006-06-07 | 2009-01-29 | Rosen Craig A | Albumin Fusion Proteins |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11555061B2 (en) | 2009-02-11 | 2023-01-17 | Albumedix, Ltd | Albumin variants and conjugates |
US10696732B2 (en) * | 2009-10-30 | 2020-06-30 | Albumedix, Ltd | Albumin variants |
US10233228B2 (en) | 2010-04-09 | 2019-03-19 | Albumedix Ltd | Albumin derivatives and variants |
US10711050B2 (en) | 2011-11-18 | 2020-07-14 | Albumedix Ltd | Variant serum albumin with improved half-life and other properties |
US10329340B2 (en) | 2012-03-16 | 2019-06-25 | Albumedix Ltd | Albumin variants |
US10501524B2 (en) | 2012-11-08 | 2019-12-10 | Albumedix Ltd | Albumin variants |
US10934341B2 (en) | 2012-11-08 | 2021-03-02 | Albumedix, Ltd. | Albumin variants |
US10633428B2 (en) | 2015-08-20 | 2020-04-28 | Albumedix Ltd | Albumin variants and conjugates |
US12116400B2 (en) | 2015-08-20 | 2024-10-15 | Sartorius Albumedix Limited | Albumin variants and conjugates |
WO2022032175A1 (en) | 2020-08-06 | 2022-02-10 | Cidara Therapeutics, Inc. | Methods for the synthesis of protein-drug conjugates |
WO2022032188A1 (en) | 2020-08-06 | 2022-02-10 | Cidara Therapeutics, Inc. | Methods for the synthesis of protein-drug conjugates |
WO2022133281A1 (en) | 2020-12-17 | 2022-06-23 | Cidara Therapeutics, Inc. | Compositions and methods for the treatment of human immunodeficiency virus |
Also Published As
Publication number | Publication date |
---|---|
WO2010092135A3 (en) | 2010-10-07 |
EP2396347B1 (en) | 2017-04-12 |
KR20110128827A (en) | 2011-11-30 |
JP5936112B2 (en) | 2016-06-15 |
EP2396347A2 (en) | 2011-12-21 |
JP2016145247A (en) | 2016-08-12 |
US9493545B2 (en) | 2016-11-15 |
CN106986933A (en) | 2017-07-28 |
EP3243835A1 (en) | 2017-11-15 |
ES2630253T3 (en) | 2017-08-18 |
JP2012517235A (en) | 2012-08-02 |
WO2010092135A2 (en) | 2010-08-19 |
US20200392206A1 (en) | 2020-12-17 |
JP6279005B2 (en) | 2018-02-14 |
DK2396347T3 (en) | 2017-07-24 |
SG172789A1 (en) | 2011-08-29 |
EP3243835B1 (en) | 2024-04-10 |
KR101722961B1 (en) | 2017-04-04 |
CN102317315A (en) | 2012-01-11 |
US11555061B2 (en) | 2023-01-17 |
US20110313133A1 (en) | 2011-12-22 |
SG2014012918A (en) | 2014-04-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11555061B2 (en) | Albumin variants and conjugates | |
US12116400B2 (en) | Albumin variants and conjugates | |
US20110124576A1 (en) | Transferrin Variants and Conjugates | |
US20190315836A1 (en) | Albumin variants | |
US10501524B2 (en) | Albumin variants | |
US20130225496A1 (en) | Albumin Variants |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ALBUMEDIX LTD, UNITED KINGDOM Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NOVOZYMES BIOPHARMA DK A/S (BI-NAME ALBUMEDIX A/S);REEL/FRAME:046093/0808 Effective date: 20180412 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: AWAITING TC RESP., ISSUE FEE NOT PAID |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE |