CN118562771A - 一种免疫球蛋白降解酶IdeE的突变体 - Google Patents
一种免疫球蛋白降解酶IdeE的突变体 Download PDFInfo
- Publication number
- CN118562771A CN118562771A CN202410654527.XA CN202410654527A CN118562771A CN 118562771 A CN118562771 A CN 118562771A CN 202410654527 A CN202410654527 A CN 202410654527A CN 118562771 A CN118562771 A CN 118562771A
- Authority
- CN
- China
- Prior art keywords
- mutant
- idee
- protein
- last
- degrading enzyme
- 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.)
- Pending
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 77
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 77
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 52
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 52
- 230000000593 degrading effect Effects 0.000 title claims abstract description 43
- 230000000694 effects Effects 0.000 claims abstract description 70
- 108090000623 proteins and genes Proteins 0.000 claims description 86
- 239000003814 drug Substances 0.000 claims description 64
- 102000004169 proteins and genes Human genes 0.000 claims description 59
- 235000018102 proteins Nutrition 0.000 claims description 57
- 235000001014 amino acid Nutrition 0.000 claims description 48
- 150000001413 amino acids Chemical group 0.000 claims description 47
- 229940079593 drug Drugs 0.000 claims description 47
- 229940024606 amino acid Drugs 0.000 claims description 43
- 241000700605 Viruses Species 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 25
- 230000035772 mutation Effects 0.000 claims description 25
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 22
- 210000004027 cell Anatomy 0.000 claims description 20
- 229930182817 methionine Natural products 0.000 claims description 19
- 229960004452 methionine Drugs 0.000 claims description 19
- 210000004369 blood Anatomy 0.000 claims description 18
- 239000008280 blood Substances 0.000 claims description 18
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 17
- 229960002885 histidine Drugs 0.000 claims description 17
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 17
- 239000013603 viral vector Substances 0.000 claims description 17
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 16
- 235000004279 alanine Nutrition 0.000 claims description 15
- 239000004475 Arginine Substances 0.000 claims description 14
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 14
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 14
- 239000004472 Lysine Substances 0.000 claims description 14
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 14
- 239000004473 Threonine Substances 0.000 claims description 14
- 229960003121 arginine Drugs 0.000 claims description 14
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 14
- 229960002989 glutamic acid Drugs 0.000 claims description 14
- 235000013922 glutamic acid Nutrition 0.000 claims description 14
- 239000004220 glutamic acid Substances 0.000 claims description 14
- 229960003646 lysine Drugs 0.000 claims description 14
- 229960002898 threonine Drugs 0.000 claims description 14
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 13
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 13
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 13
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 13
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 13
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 13
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 13
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 13
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 13
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 13
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 13
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 13
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 13
- 229960001230 asparagine Drugs 0.000 claims description 13
- 235000009582 asparagine Nutrition 0.000 claims description 13
- 229960005261 aspartic acid Drugs 0.000 claims description 13
- 235000003704 aspartic acid Nutrition 0.000 claims description 13
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 13
- 229960000310 isoleucine Drugs 0.000 claims description 13
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 13
- 229960003136 leucine Drugs 0.000 claims description 13
- 229960001153 serine Drugs 0.000 claims description 13
- 229960004799 tryptophan Drugs 0.000 claims description 13
- 229960004441 tyrosine Drugs 0.000 claims description 13
- 235000002374 tyrosine Nutrition 0.000 claims description 13
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 13
- 229960004295 valine Drugs 0.000 claims description 13
- 239000004474 valine Substances 0.000 claims description 13
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 12
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 12
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 12
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 12
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 12
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 12
- 229960002433 cysteine Drugs 0.000 claims description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 12
- 235000018417 cysteine Nutrition 0.000 claims description 12
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 12
- 229960002743 glutamine Drugs 0.000 claims description 12
- 229960005190 phenylalanine Drugs 0.000 claims description 12
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 12
- 235000008729 phenylalanine Nutrition 0.000 claims description 12
- 229960002429 proline Drugs 0.000 claims description 12
- 239000004471 Glycine Substances 0.000 claims description 11
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 claims description 11
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 claims description 11
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 11
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 11
- 229960002449 glycine Drugs 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 claims description 10
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 claims description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 10
- 238000001415 gene therapy Methods 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 244000309459 oncolytic virus Species 0.000 claims description 10
- 210000004899 c-terminal region Anatomy 0.000 claims description 9
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 8
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 8
- 229960003767 alanine Drugs 0.000 claims description 8
- 230000028327 secretion Effects 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims description 7
- 239000003937 drug carrier Substances 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 7
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 6
- 102000037982 Immune checkpoint proteins Human genes 0.000 claims description 5
- 108091008036 Immune checkpoint proteins Proteins 0.000 claims description 5
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 5
- 102000004127 Cytokines Human genes 0.000 claims description 4
- 108090000695 Cytokines Proteins 0.000 claims description 4
- 230000015556 catabolic process Effects 0.000 claims description 4
- 238000006731 degradation reaction Methods 0.000 claims description 4
- 229940023147 viral vector vaccine Drugs 0.000 claims description 4
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 108010052285 Membrane Proteins Proteins 0.000 claims description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 3
- 229940088597 hormone Drugs 0.000 claims description 3
- 239000005556 hormone Substances 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 101150050927 Fcgrt gene Proteins 0.000 claims description 2
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 25
- 238000003776 cleavage reaction Methods 0.000 description 105
- 230000007017 scission Effects 0.000 description 97
- 229940088598 enzyme Drugs 0.000 description 56
- 239000000047 product Substances 0.000 description 42
- 229940027941 immunoglobulin g Drugs 0.000 description 41
- 210000002966 serum Anatomy 0.000 description 33
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 30
- 102000004196 processed proteins & peptides Human genes 0.000 description 19
- 108090000765 processed proteins & peptides Proteins 0.000 description 19
- 239000000758 substrate Substances 0.000 description 19
- 229920001184 polypeptide Polymers 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 17
- 238000012217 deletion Methods 0.000 description 17
- 230000037430 deletion Effects 0.000 description 17
- 229960000575 trastuzumab Drugs 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 238000001962 electrophoresis Methods 0.000 description 14
- 239000012160 loading buffer Substances 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 230000008685 targeting Effects 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 10
- 229960003444 immunosuppressant agent Drugs 0.000 description 10
- 239000003018 immunosuppressive agent Substances 0.000 description 10
- 108091033319 polynucleotide Proteins 0.000 description 9
- 102000040430 polynucleotide Human genes 0.000 description 9
- 239000002157 polynucleotide Substances 0.000 description 9
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 8
- 108010021466 Mutant Proteins Proteins 0.000 description 8
- 102000008300 Mutant Proteins Human genes 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 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 8
- 239000013612 plasmid Substances 0.000 description 8
- 229940124597 therapeutic agent Drugs 0.000 description 8
- 201000010099 disease Diseases 0.000 description 7
- 230000001861 immunosuppressant effect Effects 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 102000038030 PI3Ks Human genes 0.000 description 6
- 108091007960 PI3Ks Proteins 0.000 description 6
- 229940079156 Proteasome inhibitor Drugs 0.000 description 6
- 241000700584 Simplexvirus Species 0.000 description 6
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 6
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 6
- 230000022131 cell cycle Effects 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 230000001973 epigenetic effect Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000003207 proteasome inhibitor Substances 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 6
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 229940045513 CTLA4 antagonist Drugs 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 5
- 229940044683 chemotherapy drug Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940126533 immune checkpoint blocker Drugs 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 102220083823 rs863224587 Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000711404 Avian avulavirus 1 Species 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 102000014150 Interferons Human genes 0.000 description 4
- 108010050904 Interferons Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 241000702263 Reovirus sp. Species 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 230000002238 attenuated effect Effects 0.000 description 4
- 229940127089 cytotoxic agent Drugs 0.000 description 4
- 239000002254 cytotoxic agent Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- -1 etc.) Proteins 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 102000003390 tumor necrosis factor Human genes 0.000 description 4
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- KGRVJHAUYBGFFP-UHFFFAOYSA-N 2,2'-Methylenebis(4-methyl-6-tert-butylphenol) Chemical compound CC(C)(C)C1=CC(C)=CC(CC=2C(=C(C=C(C)C=2)C(C)(C)C)O)=C1O KGRVJHAUYBGFFP-UHFFFAOYSA-N 0.000 description 3
- 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 3
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000828537 Homo sapiens Synaptic functional regulator FMR1 Proteins 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 241000125945 Protoparvovirus Species 0.000 description 3
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 3
- 241000193996 Streptococcus pyogenes Species 0.000 description 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 102100023532 Synaptic functional regulator FMR1 Human genes 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 3
- 108010009583 Transforming Growth Factors Proteins 0.000 description 3
- 102000009618 Transforming Growth Factors Human genes 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 229960001467 bortezomib Drugs 0.000 description 3
- 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 3
- 201000011510 cancer Diseases 0.000 description 3
- 229960004397 cyclophosphamide Drugs 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 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 3
- 229960005277 gemcitabine Drugs 0.000 description 3
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 3
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 102000006495 integrins Human genes 0.000 description 3
- 108010044426 integrins Proteins 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 229960001156 mitoxantrone 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
- 239000000178 monomer Substances 0.000 description 3
- 229960000688 pomalidomide Drugs 0.000 description 3
- UVSMNLNDYGZFPF-UHFFFAOYSA-N pomalidomide Chemical compound O=C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O UVSMNLNDYGZFPF-UHFFFAOYSA-N 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 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 3
- 229960001796 sunitinib Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229960004964 temozolomide Drugs 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229960003433 thalidomide Drugs 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 241000710929 Alphavirus Species 0.000 description 2
- 101710095342 Apolipoprotein B Proteins 0.000 description 2
- 102100040202 Apolipoprotein B-100 Human genes 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 2
- 108010029697 CD40 Ligand Proteins 0.000 description 2
- 102100032937 CD40 ligand Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 206010008025 Cerebellar ataxia Diseases 0.000 description 2
- 241000709687 Coxsackievirus Species 0.000 description 2
- 241000709675 Coxsackievirus B3 Species 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- 108010086800 Glucose-6-Phosphatase Proteins 0.000 description 2
- 102000003638 Glucose-6-Phosphatase Human genes 0.000 description 2
- 102000004547 Glucosylceramidase Human genes 0.000 description 2
- 108010017544 Glucosylceramidase Proteins 0.000 description 2
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 2
- 101710083479 Hepatitis A virus cellular receptor 2 homolog Proteins 0.000 description 2
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 101000709472 Homo sapiens Sialic acid-binding Ig-like lectin 15 Proteins 0.000 description 2
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 2
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 2
- 101000904724 Homo sapiens Transmembrane glycoprotein NMB Proteins 0.000 description 2
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 2
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 2
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- 102000013462 Interleukin-12 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 102000017578 LAG3 Human genes 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 241000711386 Mumps virus Species 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000702244 Orthoreovirus Species 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 241000709664 Picornaviridae Species 0.000 description 2
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 102100034361 Sialic acid-binding Ig-like lectin 15 Human genes 0.000 description 2
- 241000194048 Streptococcus equi Species 0.000 description 2
- 102000008221 Superoxide Dismutase-1 Human genes 0.000 description 2
- 108010021188 Superoxide Dismutase-1 Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 2
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- 108020004440 Thymidine kinase Proteins 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 2
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 2
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 241000711975 Vesicular stomatitis virus Species 0.000 description 2
- 241000726445 Viroids Species 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 102200129340 c.176C>T Human genes 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229940117681 interleukin-12 Drugs 0.000 description 2
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 108010068617 neonatal Fc receptor Proteins 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 229960005547 pelareorep Drugs 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 102200095430 rs28940294 Human genes 0.000 description 2
- 102220053185 rs727504747 Human genes 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- SSOORFWOBGFTHL-OTEJMHTDSA-N (4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-5-carbamimidamido-1-[[(2S)-5-carbamimidamido-1-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-diaminohexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SSOORFWOBGFTHL-OTEJMHTDSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-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
- 101150033426 2.5 gene Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 101150096316 5 gene Proteins 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 102100024378 AF4/FMR2 family member 2 Human genes 0.000 description 1
- 101710184468 AF4/FMR2 family member 2 Proteins 0.000 description 1
- 102100036664 Adenosine deaminase Human genes 0.000 description 1
- 102000002572 Alpha-Globulins Human genes 0.000 description 1
- 108010068307 Alpha-Globulins Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102100032187 Androgen receptor Human genes 0.000 description 1
- 102100022987 Angiogenin Human genes 0.000 description 1
- 102400000068 Angiostatin Human genes 0.000 description 1
- 108010079709 Angiostatins Proteins 0.000 description 1
- 102100026565 Ataxin-8 Human genes 0.000 description 1
- 101710147490 Ataxin-8 Proteins 0.000 description 1
- 102000014461 Ataxins Human genes 0.000 description 1
- 108010078286 Ataxins Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 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
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102000006734 Beta-Globulins Human genes 0.000 description 1
- 108010087504 Beta-Globulins Proteins 0.000 description 1
- 101710124976 Beta-hexosaminidase A Proteins 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 1
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 1
- BHKSYEZGBQDNRW-SCGRZTRASA-N C(CCC(=O)O)(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O Chemical compound C(CCC(=O)O)(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O BHKSYEZGBQDNRW-SCGRZTRASA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 102100031168 CCN family member 2 Human genes 0.000 description 1
- 102000049320 CD36 Human genes 0.000 description 1
- 108010045374 CD36 Antigens Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 101100123850 Caenorhabditis elegans her-1 gene Proteins 0.000 description 1
- 108090000312 Calcium Channels Proteins 0.000 description 1
- 102000003922 Calcium Channels Human genes 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 1
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 102100023804 Coagulation factor VII Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 1
- 102000012437 Copper-Transporting ATPases Human genes 0.000 description 1
- 108010022637 Copper-Transporting ATPases Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 101710095468 Cyclase Proteins 0.000 description 1
- 102000005927 Cysteine Proteases Human genes 0.000 description 1
- 108010005843 Cysteine Proteases Proteins 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 208000014094 Dystonic disease Diseases 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 102220542356 Endogenous retrovirus group K member 113 Pro protein_T24A_mutation Human genes 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 108010055196 EphA2 Receptor Proteins 0.000 description 1
- 108010055334 EphB2 Receptor Proteins 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 102100031968 Ephrin type-B receptor 2 Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 229940122601 Esterase inhibitor Drugs 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
- 108010054265 Factor VIIa Proteins 0.000 description 1
- 108010014173 Factor X Proteins 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 102100035139 Folate receptor alpha Human genes 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 208000001914 Fragile X syndrome Diseases 0.000 description 1
- 102000003869 Frataxin Human genes 0.000 description 1
- 108090000217 Frataxin Proteins 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 1
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 102100032530 Glypican-3 Human genes 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 239000000095 Growth Hormone-Releasing Hormone Substances 0.000 description 1
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 102100030595 HLA class II histocompatibility antigen gamma chain Human genes 0.000 description 1
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 102000002268 Hexosaminidases Human genes 0.000 description 1
- 108010000540 Hexosaminidases Proteins 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101000873082 Homo sapiens Ataxin-1 Proteins 0.000 description 1
- 101000895114 Homo sapiens Ataxin-2 Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101001023230 Homo sapiens Folate receptor alpha Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 description 1
- 101001082627 Homo sapiens HLA class II histocompatibility antigen gamma chain Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 1
- 101000961414 Homo sapiens Membrane cofactor protein Proteins 0.000 description 1
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101001098868 Homo sapiens Proprotein convertase subtilisin/kexin type 9 Proteins 0.000 description 1
- 101000834948 Homo sapiens Tomoregulin-2 Proteins 0.000 description 1
- 101000662686 Homo sapiens Torsin-1A Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 101000760175 Homo sapiens Zinc finger protein 35 Proteins 0.000 description 1
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- 102000000853 LDL receptors Human genes 0.000 description 1
- 108010001831 LDL receptors Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 1
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 208000015439 Lysosomal storage disease Diseases 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 102100039373 Membrane cofactor protein Human genes 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- 108090000015 Mesothelin Proteins 0.000 description 1
- 102000003735 Mesothelin Human genes 0.000 description 1
- 102000014962 Monocyte Chemoattractant Proteins Human genes 0.000 description 1
- 108010064136 Monocyte Chemoattractant Proteins Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 1
- 101100369076 Mus musculus Tdgf1 gene Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 206010068871 Myotonic dystrophy Diseases 0.000 description 1
- 206010028885 Necrotising fasciitis Diseases 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 108010074223 Netrin-1 Proteins 0.000 description 1
- 102000009065 Netrin-1 Human genes 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 102100029268 Neurotrophin-3 Human genes 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000007981 Ornithine carbamoyltransferase Human genes 0.000 description 1
- 101710198224 Ornithine carbamoyltransferase, mitochondrial Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 102100038955 Proprotein convertase subtilisin/kexin type 9 Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 1
- 102100031176 Retinoid isomerohydrolase Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 102100036546 Salivary acidic proline-rich phosphoprotein 1/2 Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 241000710961 Semliki Forest virus Species 0.000 description 1
- 241000837158 Senecavirus A Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 101710189648 Serine/threonine-protein phosphatase Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102100022831 Somatoliberin Human genes 0.000 description 1
- 101710142969 Somatoliberin Proteins 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 108010061312 Sphingomyelin Phosphodiesterase Proteins 0.000 description 1
- 102000011971 Sphingomyelin Phosphodiesterase Human genes 0.000 description 1
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 1
- 206010061372 Streptococcal infection Diseases 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241001505901 Streptococcus sp. 'group A' Species 0.000 description 1
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 1
- 102000006467 TATA-Box Binding Protein Human genes 0.000 description 1
- 108010044281 TATA-Box Binding Protein Proteins 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108010041111 Thrombopoietin Proteins 0.000 description 1
- 102000036693 Thrombopoietin Human genes 0.000 description 1
- 102100026160 Tomoregulin-2 Human genes 0.000 description 1
- 102100037454 Torsin-1A Human genes 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 102000004903 Troponin Human genes 0.000 description 1
- 108090001027 Troponin Proteins 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 241000710886 West Nile virus Species 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 102100024672 Zinc finger protein 35 Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 108010080146 androgen receptors Proteins 0.000 description 1
- 108010072788 angiogenin Proteins 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
- 201000004562 autosomal dominant cerebellar ataxia Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction 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
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 102220352428 c.92G>A Human genes 0.000 description 1
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 241001492478 dsDNA viruses, no RNA stage Species 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 229950002830 enadenotucirev Drugs 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 108060002566 ephrin Proteins 0.000 description 1
- 102000012803 ephrin Human genes 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 239000002329 esterase inhibitor Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229940012413 factor vii Drugs 0.000 description 1
- 229940012414 factor viia 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
- 108700014844 flt3 ligand Proteins 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000010353 genetic engineering Methods 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
- 108010070004 glucose receptor Proteins 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 102000056417 human ATXN1 Human genes 0.000 description 1
- 102000056418 human ATXN2 Human genes 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 208000009091 myxoma Diseases 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 201000007970 necrotizing fasciitis Diseases 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 108010081726 netrin-2 Proteins 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 229940056106 nipocalimab Drugs 0.000 description 1
- 229940121468 nirsevimab Drugs 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical class NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 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 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000030925 respiratory syncytial virus infectious disease Diseases 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 108010054126 retinoid isomerohydrolase Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 229950005039 rozanolixizumab Drugs 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 108010025625 vocimagene amiretrorepvec Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- 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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/2201—Streptopain (3.4.22.10)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种免疫球蛋白降解酶IdeE的突变体。所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列;至少对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体;所述的突变体的功能至少包含所述免疫球蛋白降解酶IdeE的功能。本发明提供的免疫球蛋白降解酶突变体的活性和热稳定性高于野生型IdeE。
Description
本发明为申请号为“CN202180013436.2”,发明名称为“一种免疫球蛋白降解酶IdeE的突变体”的发明专利的分案申请
技术领域
本发明涉及生物技术领域,具体涉及一种免疫球蛋白的降解酶的突变体。
背景技术
化脓链球菌是人畜常见的病原菌之一,广泛存在于自然界及人或动物的口咽腔、呼吸道及肠道中。链球菌感染会引发相关疾病,较轻的病症如化脓性皮炎、咽炎,比较严重的疾病如败血症、坏死性筋膜炎和中毒性休克综合症。来自酿脓链球菌的免疫球蛋白G降解酶(Immunoglobulin G-degrading enzyme of Streptococcus pyogenes,IdeS),是一种常见的A型链球菌(Group A Streptococcus pyogenes,GAS)半胱氨酸蛋白酶,具有水解IgG的肽链内肽酶活性(Agniswamy J,Lei B,Musser J M等,J Biol Chem,2004,279:52789-52796.Lei B,DeLeo F R,Reid SD等,Infect Immun,2002,70:6880-6890.Von Pawel-Rammingen U,Johansson B P,Bjorck L..EMBO J,2002,21:1607-1615.)。作为一种致病菌的毒力因子,它可以识别抗体下铰链区CH1和CH2结构域并特异性降解IgG,获得同质的F(ab)2和Fc片段,帮助GAS逃避抗体介导的吞噬及细胞毒作用,从而减弱宿主免疫系统对GAS的杀伤(Von Pawel-Rammingen U.J Innate Immunity,2012,4:132-140.Su,Y.-F.等,Molecular Immunology,2011,49:134-142.)。
免疫球蛋白G(Immunoglobulin G,IgG),是血清主要的抗体成分,约占血清免疫球蛋白的75%,在机体免疫中主要起保护作用,能有效预防感染性疾病。除了具有保护性作用之外,IgG也和疾病相关。在一些自身免疫性疾病中,IgG抗体与人体自身分子发生反应,在器官移植中IgG会引起急性移植排斥反应。IdeS特异性降解IgG,从而使IgG失去其应有功能从而实现免疫抑制。
目前应用于临床的IdeS存在活性不佳,人体预存抗体高的问题。IdeS是人类病原菌的毒力因子,临床研究发现正常人在正常生理条件下接近100%检出抗IdeS抗体,导致IdeS的使用方式给药效率不高,同时存在安全性问题。
与IdeS序列同源性为70%左右的IdeE蛋白酶来源于马链球菌兽疫亚种Streptococcus equi ssp.equi,是一种马的致病菌(Jonas Bengt Guss.FEMSMicrobiol Lett,2006,262:230-235)。IdeE与IdeS这两种酶在完全相同的位置切断IgG,切割具有高度可重复性和特异性,并且有着非常类似的底物范围。优于IdeE来自马致病菌,推测其在人体内预先存在的抗体可能远低于IdeS,更适合开发用于治疗和预防由IgG抗体介导疾病的免疫抑制剂。但野生型IdeE和IdeS一样,也存在活性低的问题。因此需要通过分子设计和突变筛选提高IdeE的活性,降低临床使用剂量,从而降低由高剂量细菌来源蛋白带来的风险。这正是本发明所要达到的目的。
发明内容
本发明的第一方面涉及免疫球蛋白降解酶IdeE的突变体,其特征在于,所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成;所述突变选自下组:
(1)对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体;和/或,
(2)对所述免疫球蛋白降解酶IdeE进行截短,删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个,前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列;和/或,
(3)对所述免疫球蛋白降解酶IdeE进行截短,删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列;
其中所述突变体具有高于所述免疫球蛋白降解酶IdeE的活性和/或热稳定性。
本发明的第二方面涉及包含本发明的突变体的蛋白。所述蛋白在所述突变体的N末端连接有分泌信号序列和/或甲硫氨酸;和/或所述蛋白在所述突变体的C末端连接有组氨酸标签。
本发明的第三-第五方面涉及一种编码本发明的突变体或蛋白的核苷酸,包含所述核苷酸的表达载体,以及包含所述表达载体或者表达本发明的突变体或蛋白的宿主细胞。
本发明的第六方面涉及一种组合物或试剂盒,其包含:本发明的突变体或蛋白;和任选的选自下组的物质:药学上可接受的载体或赋形剂、抗体或者含有Fc的蛋白、病毒载体药物。
附图说明
图1为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图2为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图3为5个N端截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图4为50℃条件下保温1h后5个N端截短突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图5为2个C截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图6为5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图7为50℃条件下保温1h后5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图8为不同浓度的E97D_del18突变体和IdeS切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图9为不同浓度的E97D_del18突变体和IdeZ切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图10为E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图11为E97D_del18突变体在小鼠和人血清中产生的切割产物SDS-PAGE凝胶电泳图。
图12A-12D为不同浓度的E97D_del18在比格犬、大鼠、小鼠、兔、猴和猪血清中切割IgG产生的切割产物SDS-PAGE凝胶电泳图。
图13为E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图14A和14B为具有不同突变组合的突变体和IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
发明详述
I.具有免疫球蛋白降解酶活性的功能性多肽
本发明的第一方面,提供了一种功能性多肽,所述功能性多肽具有免疫球蛋白降解酶的活性并包含有基于SEQ ID NO:2所示的氨基酸序列的突变体,所述突变体选自:
(1)在SEQ ID NO:2的第8、10、24、59、97和280中的一位或者多位氨基酸进行替换后得到突变体;和/或,
(2)SEQ ID NO:2的N端的截短突变体,选自删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个、前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列;和/或,
(3)SEQ ID NO:2的C端的截短突变体,选自删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列。
本发明所述的突变体具有所述免疫球蛋白降解酶IdeE的功能,优选地还具有提高的IgG切割活性和热稳定性。
本发明中的术语“具有高于免疫球蛋白降解酶IdeE的活性”指的是所述突变体降解免疫球蛋白的能力优于野生型免疫球蛋白降解酶IdeE。
本发明中的术语“比IdeE具有更高的热稳定性“指的是所述突变体在某温度下维持一段时间后,降解免疫球蛋白的能力优于同等条件下的野生型免疫球蛋白降解酶IdeE。
本发明所述的突变体优选通过基因工程重组方式生产得到。
优选地,所述突变体与SEQ ID NO:2所示序列具有至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的同一性。
更优选地,对所述位置8、10、24、59、97或者280的氨基酸进行替换,例如所得突变体的氨基酸序列如SEQ ID NO:3~17、SEQ ID NO:35中任一所示;
或者删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸,所得突变体的氨基酸序列如SEQ ID NO:18~22中任一所示;
或者删除所述免疫球蛋白降解酶IdeE C端的最后1个、最后5个、最后8个或者最后10个氨基酸,例如所得突变体的氨基酸序列如SEQ ID NO:23~24中任一所示;
或者对所述位置8、10、24、59、97或者280进行替换并同时删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸,优选删除前18个氨基酸,例如所得突变体的氨基酸序列如SEQ ID NO:25~29中任一所示。
或者对所述位置8、10、24、59、97或者280进行替换并同时删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸并同时删除所述免疫球蛋白降解酶IdeE C端的最后1个、最后5个、最后8个或者最后10个氨基酸,优选删除前18个氨基酸,优选删除后5个氨基酸,例如所得突变体的氨基酸序列如SEQ ID NO:30~34中任一所示。
在本发明一个优选的实施方案中,所述氨基酸的替换选自下组:
(1)SEQ ID NO:2的位置8处的苏氨酸被替换成半胱氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、谷氨酸、丙氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、丝氨酸、缬氨酸、精氨酸和赖氨酸中的任意一种;
(2)SEQ ID NO:2的位置10处的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(3)SEQ ID NO:2的位置24处的苏氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、天冬酰胺、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(4)SEQ ID NO:2的位置59处的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(5)SEQ ID NO:2的位置97处的谷氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(6)SEQ ID NO:2的位置280处的精氨酸被替换成丙氨酸、天冬氨酸、谷氨酸、半胱氨酸、丝氨酸、苯丙氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种。
在本发明一个更优选的实施方案中,所述氨基酸的替换选自下组:
(1)SEQ ID NO:2的位置8处的苏氨酸被替换成天冬氨酸、谷氨酸、色氨酸或酪氨酸;
(2)SEQ ID NO:2的位置10处的丙氨酸被替换成赖氨酸或精氨酸;
(3)SEQ ID NO:2的位置24处的苏氨酸被替换成丙氨酸、甘氨酸或丝氨酸;
(4)SEQ ID NO:2的位置59处的丙氨酸被替换成异亮氨酸、亮氨酸或缬氨酸;
(5)SEQ ID NO:2的位置97处的谷氨酸被替换成天冬酰胺;和/或
(6)SEQ ID NO:2的位置280处的精氨酸被替换成组氨酸或赖氨酸。
在另一优选的实施方案中,基于SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:14、SEQID NO:1、SEQ ID NO:5和SEQ ID NO:16这5个经氨基酸替换得到的序列进一步删除其N端的前18个氨基酸,所得突变体的氨基酸序列如序列表中SEQ ID NO:25~29所示。
在另一优选的实施方案中,基于SEQ ID NO:26~29这5个经氨基酸替换得到的序列进一步删除其C端的5个或10氨基酸,所得突变体的氨基酸序列如序列表中SEQ ID NO:30~34所示。
在另一优选的实施方案中,基于SEQ ID NO:14~16这3个突变体进一步进行组合突变,所得突变体的氨基酸序列如序列表中SEQ ID NO:35所示。在另一优选的实施方案中,基于SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:1、SEQ ID NO:5和SEQ ID NO:16这5个经氨基酸替换得到的序列进一步删除其N端的前18个氨基酸,所得突变体的氨基酸序列如序列表中SEQ ID NO:25~29所示。
优选地,还可以对本发明中所述的突变体进行进一步的突变,进一步突变后所得的变体的序列与SEQ ID NO:2的序列具有至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%的同一性,同时具有免疫球蛋白降解酶IdeE的功能。
本发明使用的IdeE全序列作为GenBank登录号ABF57910.1是公众可获得的,其序列在本文中作为SEQ ID NO:1提供。该序列包括N末端甲硫氨酸,接着是33个氨基酸的分泌信号序列,接着是IdeE编码序列。N末端甲硫氨酸和信号序列通常被去除以形成成熟的IdeE蛋白,其序列在本文中作为SEQ ID NO:2提供。除非另有说明,对本文公开的免疫球蛋白降解酶序列中氨基酸位置的编号的所有提及均基于从N末端开始的SEQ ID NO:2中相应位置的编号。
本发明还提供一种包含如上所述的突变体的蛋白。
在一个优选的实施例中,所述蛋白为在上述突变体的N末端包含信号肽;优选地,所述蛋白在所述突变体的N末端连接有分泌信号序列并在所述分泌序列的N末端连接有甲硫氨酸和/或在所述突变体的C末端连接有组氨酸标签;更优选地,所述蛋白从N末端至C末端包含如下或由如下组成:甲硫氨酸、分泌信号序列和所述突变体。
本发明的第二方面,提供了一种编码如上所述的蛋白或者突变体的核苷酸。
本发明还提供一种包含所述核苷酸的表达载体。
本发明还提供一种包含如上所述的表达载体的宿主细胞,或者表达如上所述的蛋白或者突变体的宿主细胞。
所述的宿主细胞可为本领域常规的用于表达蛋白或者多肽的细胞,选自大肠杆菌细胞、酵母细胞等。
II.药物组合
本发明的第三方面,提供了一种组合物,其包括免疫球蛋白降解酶或其突变体或者包含免疫球蛋白降解酶或其突变体的蛋白,以及任选的药学上可接受的载体或赋形剂。在一个具体的实施方案中,所述免疫球蛋白降解酶选自IdeE、IdeS和IdeZ。在一个具体的实施方案中,所述免疫球蛋白降解酶的突变体为如上所述的突变体,所述蛋白为包含如上所述的突变体的蛋白。在一个具体的实施方案中,本发明的组合物进一步包含:抗体或者含有Fc的蛋白。在一个具体的实施方案中,所述抗体的靶点选自下组:细胞表面蛋白、细胞因子、激素、酶、胞内信使、胞间信使和免疫检查点。在一个具体的实施方案中,本发明的组合物进一步包含:病毒载体药物,优选地,所述病毒载体药物选自下组:溶瘤病毒、基因治疗病毒和病毒载体疫苗。在一个具体的实施方案中,本发明的组合物进一步包含:能降低血液IgG水平的药物,优选的,所述降低血液IgG水平的药物选自下组:FcRn抗体、与FcRn高亲和力的Fc片段变体。
2.1抗体的靶点
优选地,如上所述的组合物,其中,所述抗体的靶点可为细胞表面蛋白,包括但不限于:AFP、αv整联蛋白(integrin)、α4β7整联蛋白、BCMA、CD2、CD3、CD19、CD20、CD22、CD25、CD30、CD32、CD33、CD36、CD40、CD46、CD52、CD56、CD64、CD70、CD74、CD79、CD80、CD86、CD105、CD121、CD123、CD133、CD138、CD174、CD205、CD227、CD326、CD340、CEA、c-Met、Cripto、CA1X、Claudin18.2、ED-B、EGFR、EpCAM、EphA2、EphB2、FAP、FOLR1、GD2、Globo H、GPC3、GPNMB、HER-1、HER-2、HER-3、MAGE-A3、Mesothelin、MUC16、GPNMB、PSMA、TMEFF2、TAG-72、5T4、ROR-1、Sca-1、SP、VEGF或WT1。
所述抗体的靶点可为细胞因子,包括但不限于:白介素IL-1至IL-13、肿瘤坏死因子α和β、干扰素α、β和γ、肿瘤生长因子β(TGF-β)、集落刺激因子(CSF)或粒细胞单核细胞集落刺激因子(GM-CSF)。见HumanCytokines:Handbook for Basic&Clinical Research(Aggrawal等编,Blackwell Scientific,Boston,MA 1991)。
所述抗体的靶点可为激素、酶、胞内和胞间信使,例如:腺苷环化酶、鸟苷环化酶或磷脂酶C。
所述抗体的靶点可为免疫检查点,所述的免疫检查点包括:CTLA-4、PD-1、PD-L1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。
2.2靶向药物
优选地,如上所述组合物,其中,所述组合物还包括靶向药物或化疗药物或免疫检查点阻断剂,所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂,所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物,所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体;所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂,所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine,所述酪氨酸激酶抑制剂例如舒尼替尼,所述免疫抑制剂例如环磷酰胺,所述蛋白酶体抑制剂例如硼替佐米,所述免疫抑制剂例如沙利度胺、泊马度胺,所述细胞毒药物例如吉西他滨、替莫唑胺,所述细胞周期非特异性药物例如米托蒽醌。
2.3能降低血液IgG水平的药物
优选地,如上所述的组合物,其中,所述能降低血液IgG水平的多肽药物能够阻断血液IgG和FcRn蛋白的结合。优选地,所述多肽与人FcRn蛋白的亲和力高于血液IgG和人FcRn蛋白的亲和力;所述IgG选自IgG1、IgG2、IgG3、IgG4。优选地,所述多肽包含抗体Fc片段变体,所述变体包含能够提高Fc和FcRn亲和力的突变,所述突变优选为YTE、YTEKF、LS、NHS,所述抗体Fc片段例如Efgartigimod。所述变体可以为单体、二聚体、多聚体。可用于本发明的所述YTE、YTEKF、LS、NHS等突变,所述突变的位置分别如Dall'Acqua等所描述(WF,D.A.等(2002).Journal of immunology(Baltimore,Md.:1950)169(9):5171-5180.)、Lee等所描述(Lee,C.H.等(2019).Nat Commun 10(1):5031.)。所述突变对象选自人IgG,所述IgG选自IgG1、IgG2、IgG3、IgG4。
可用于本发明的其他Fc片段变体,所述变体包含包括但不限于Dall'Acqua等描述的突变(WF,D.A.等(2002).Journal of immunology(Baltimore,Md.:1950)169(9):5171-5180.)、Shan等描述的突变(Shan,L.等(2016).PLoS One 11(8):e0160345.)、Lee等描述的突变(Lee,C.H.等(2019).Nat Commun 10(1):5031.)、Mackness等描述的突变(Mackness,B.C.等(2019).MAbs 11(7):1276-1288.)、Christophe等描述的突变(Dumet Christophe,Pottier Jérémy,Gouilleux-Gruart Valérie等,MAbs,2019,11:1341-1350.)。
优选地,所述多肽包含抗体Fc片段变体,所述变体包含能够提高Fc和FcγR亲和力的突变,所述变体优选为S239D/I322E、S239D/I322E/A330L、K326W/E333S、R214K突变;所述变体优选为无岩藻糖修饰。所述变体可以为单体、二聚体、多聚体。可用于本发明的其他Fc片段变体,所述变体包含包括但不限于Wang等描述的突变(Wang Xinhua.,Mathieu Mary.,Brezski Randall J.(2018).Protein Cell,9(1),63-73.doi:10.1007/s13238-017-0473-8)。
优选地,所述包含能够提高Fc和FcRn亲和力的变体同时包含能够提高Fc和FcγR亲和力的突变。所述变体可以为单体、二聚体、多聚体。
优选地,如上所述的药物组合,其中,所述多肽选自抗FcRn抗体,所述抗体例如Nipocalimab、Rozanolixizumab、RVT-1401、HBM9161、ALXN1830、SYNT001、Nirsevimab。
优选地,如上所述的药物组合,其中,所述多肽选自能特异性结合FcRn的小肽片段,所述小肽片段的长度为10-70个氨基酸;所述小肽片段例如ABY-039。
优选地,所述多肽选自能特异性结合FcRn的Fc多聚体,所述Fc多聚体例如GL-2045、M230、PRIM、HexaGardTM、CSL777、Hexavalent molecules by UCB。
优选地,所述多肽包括但不限于Sockolosky等描述的多肽片段(SockoloskyJonathan T,Szoka Francis C.Adv.Drug Deliv.Rev.,2015,91:109-24)。
2.4病毒载体药物
优选地,如上所述的组合物,其中,所述病毒载体药物中,所述病毒载体药物所用的病毒选自ssDNA类病毒、dsDNA类病毒、ssRNA类病毒或dsRNA类病毒;和/或,所述病毒载体药物所用的病毒选自野生型病毒株或自然减毒株、基因工程选择性减毒株、基因加载型病毒株、基因转录靶向型病毒株。
优选地,所述野生型病毒株或自然减毒株选自新城疫病毒、呼肠孤病毒、流行性腮腺炎病毒、西尼罗河病毒、腺病毒、牛痘病毒等。
优选地,所述基因工程选择性减毒株通过人工方式删除关键基因而实现病毒复制的肿瘤选择性,例如胸苷激酶(Thymidinekinase,TK)敲除的基因改造人单纯疱疹病毒I(HSV-1),所述基因工程选择性减毒株例如ONYX-015、G207。ONYX-015在E1b区域删除了827bp,并且在针对E1B55K蛋白的基因进行点突变,使其表达基因提前终止,无法表达E1B55K蛋白。G207删除了γ34.5基因,该基因为HSV-1的神经毒性决定因素。
优选地,所述基因加载型病毒株加载了外源基因,所述外源基因例如为粒细胞巨噬细胞集落刺激因子(GM-CSF),所述基因加载型病毒株例如为JX-594或T-VEC。
优选地,所述基因转录靶向型病毒株,即在病毒必需基因前插入组织或肿瘤特异性启动子来控制溶瘤病毒在肿瘤细胞内复制,所述基因转录靶向型病毒株例如为G92A。
优选地,如上所述的药物组合,其中,所述ssDNA类病毒选自细小病毒(parvovirus),优选所述细小病毒为H-1PV病毒。
优选地,所述dsDNA类病毒选自单纯疱疹病毒(herpes simplex virus)、腺病毒(adeno virus)、poxvirus;更优选地,所述单纯疱疹病毒优选为I型单纯疱疹病毒HSV-1,例如为R3616、T-VEC、HF10、G207、NV1020、OrienX010,所述poxvirus选自Pexa-Vec(vacciniaviruse)、JX-594(vaccinia viruse)、GL-ONC1、Myxoma;所述腺病毒选自Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301)。
优选地,所述ssRNA类病毒选自Picornavirus、alphavirus、Retroviruses、Paramyxoviruses、Rhabdoviruses;优选地,所述Picornavirus选自CAVATAK、PVS-RIPO、CVA21(enterovirus)、RIGVIR,所述alphavirus选自M1、Sindbis AR339、Semliki Forestvirus,所述Retroviruses选自Toca511,所述Paramyxoviruses选自MV-NIS、PV701(Newcastledisease virus),所述Rhabdoviruses选自VSV-IFNβ、MG1-MAGEA3、VSV-GP。
优选地,所述dsRNA类病毒选自Reoviruses;优选地,所述Reoviruses选自Pelareorep、呼肠孤病毒(Reolysin)、牛痘病毒(vaccinia virus)、腮腺炎病毒、人类免疫缺陷病毒(human immunodeficiency virus,HIV);优选的,所述RNA类病毒选自呼肠孤病毒(reovirus)、柯萨奇病毒(coxsackievirus)、脊髓灰质炎病毒(polio virus)、猪塞内加谷病毒(seneca valley virus)、麻疹病毒(measles virus)、新城疫病毒(newcastlediseasevirus)、水泡性口炎病毒(vesicular stomatitis virus)、流感病毒。
优选地,如上所述的药物组合,其中,所述溶瘤病毒表达外源基因,所述外源基因优选双特异性T细胞结合子(Bispecific T cell engagers,BiTE)、scFv片段、细胞因子、趋化因子。所述BiTE能结合CD3等激活T细胞的分子,同时能结合癌细胞表面的抗原靶点;所述scFv靶向免疫检查点;所述免疫检查点包括CTLA-4、PD-1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA。所述细胞因子、趋化因子例如GM-CSF、白细胞介素-2(IL-2)、白细胞介素-12(IL-12)、干扰素(IFN)、肿瘤坏死因子(TNF)、可溶性CD80、CCL3。
2.5基因治疗药物
优选地,如上所述的组合物,其中,所述基因治疗病毒表达外源基因,所述外源基因编码基因缺陷疾病所需的蛋白,所述蛋白选自酸性α-葡糖苷酶、铜转运ATPase2、α半乳糖苷酶、精氨酸琥珀酸酯合酶、β-葡萄糖脑苷脂酶、β-己糖胺酶A、Cl蛋白酶抑制剂或Cl酯酶抑制剂、葡萄糖6磷酸酶、胰岛素、胰高血糖素、生长激素、甲状旁腺激素、生长激素释放因子、卵泡刺激素、黄体生成激素、人绒毛膜促性腺激素、血管内皮生长因子、血管生成素、血管生成抑制素、粒细胞集落刺激因子、促红细胞生成素、结缔组织生长因子、碱性成纤维细胞生长因子、酸性成纤维细胞生长因子、表皮生长因子、转化生长因子a、血小板衍生生长因子、胰岛素生长因子I和II、TGF、骨形态发生蛋白、神经生长因子、脑源性神经营养因子、神经营养蛋白NT-3和NT4/5、睫状神经营养因子、神经胶质细胞系衍生神经营养因子、神经营养素、凝集素、netrin-1和netrin-2、肝细胞生长因子、ephrins、酪氨酸羟化酶、血小板生成素、白介素(IL-1至IL-36等)、单核细胞趋化蛋白、白血病抑制因子、粒细胞巨噬细胞的蛋白质集落刺激因子、Fas配体、肿瘤坏死因子a和b、干扰素a/b/g、干细胞因子、flk-2/flt3配体、IgM,IgA,IgD和IgE、嵌合免疫球蛋白、人源化抗体、单个链抗体、T细胞受体、嵌合T细胞受体、单链T细胞受体、I类和II类MHC分子、囊性纤维化跨膜调节蛋白、凝血(凝血)因子(因子XIII,因子IX,因子VIII,因子X,因子VII,因子VIIa,蛋白C等)、视网膜色素上皮特异性65kDa蛋白、LDL受体、脂蛋白脂肪酶、鸟氨酸转氨甲酰酶、β-球蛋白、α-球蛋白、血影蛋白、α-抗胰蛋白酶腺苷脱氨酶、金属转运蛋白(ATP7A或ATP7)、磺酰胺酶、参与溶酶体贮积病的酶(ARSA)、次黄嘌呤鸟嘌呤磷酸核糖基转移酶、b-25葡糖脑苷脂酶、鞘磷脂酶、溶酶体己糖胺酶、支链酮酸脱氢酶。
优选地,如上所述的组合物,其中,所述基因治疗病毒携带外源基因,所述外源基因编码选自siRNA,反义分子,miRNA,RNAi,核酶和shRNA的抑制性核酸。所述抑制性核酸结合至多核苷酸重复疾病相关的基因,该基因的转录物或该基因的转录物的多核苷酸重复。所述疾病基因编码相关蛋白,所述蛋白选自选自亨廷顿蛋白(HTT)、脊髓球肌萎缩症X染色体上的雄激素受体、人Ataxin-1/-2/-3/-7、Cav2.1P/Q电压依赖性钙通道(CACNA1A)、TATA结合蛋白、Ataxin8反向链(ATXN80S)、丝氨酸/苏氨酸蛋白磷酸酶2A55kDa脊髓小脑性共济失调的亚型B亚型β亚型(1、2、3、6、7、8、1217型)、FMR1(脆性X综合征的脆弱性1)、脆性X相关性震颤/共济失调综合征的FMR1(脆性X智力障碍1)、脆性XE智力低下的FMR1(脆性X智力障碍2)或AF4/FMR2家庭成员2;肌强直性营养不良中的肌钙蛋白激酶(MT-PK)、Frataxin。所述疾病基因选自,超氧化物歧化酶1(SOD1)基因的突变体、与帕金森氏病和/或阿尔茨海默氏病发病机理有关的基因、载脂蛋白B(APOB)、PCSK9、HIV感染相关基因(HIVTat、TAR、HIVTAR、CCR5)、流感病毒感染中的甲型流感病毒基因组/基因序列、SARS感染中的严重急性呼吸综合征(SARS)冠状病毒基因组/基因序列、呼吸道合胞病毒感染中的呼吸道合胞病毒基因组/基因序列、埃博拉病毒感染中的埃博拉病毒基因组/基因序列、乙型和丙型肝炎病毒在乙型和丙型肝炎病毒中的基因组/基因序列、HSV感染的单纯疱疹病毒(HSV)基因组/基因序列、柯萨奇病毒B3感染的柯萨奇病毒B3基因组/基因序列、沉默原发性肌张力障碍中基因的致病性等位基因(等位基因特异性沉默)如torsinA、在移植中特异性泛I类和HLA等位基因、常染色体显性遗传性视网膜色素变性中的突变和视紫红质基因。
III.产品
本发明还提供了一种产品,所述产品含有如上所述的突变体或者蛋白和治疗剂;所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物。
本发明还提供了一种试剂盒或套装药盒,所述试剂盒包含:1)治疗有效量的包含如上所述的突变体的药物;和2)治疗有效量的治疗剂;所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物;所述病毒载体药物优选溶瘤病毒、基因治疗病毒。所述试剂盒还可以包括3)靶向药物或化疗药物或免疫检查点阻断剂。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂,所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物,所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体;所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂,所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine,所述酪氨酸激酶抑制剂例如舒尼替尼,所述免疫抑制剂例如环磷酰胺,所述蛋白酶体抑制剂例如硼替佐米,所述免疫抑制剂例如沙利度胺、泊马度胺,所述细胞毒药物例如吉西他滨、替莫唑胺,所述细胞周期非特异性药物例如米托蒽醌。
所述试剂盒或套装药盒包括药盒A和药盒B,所述药盒A包括治疗有效量的如上所述突变体或蛋白,所述药盒B包括治疗有效量的治疗剂;所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物;所述病毒载体药物优选溶瘤病毒、基因治疗病毒。所述套装药盒还可以包括药盒C。所述药盒C包括靶向药物或化疗药物或免疫检查点阻断剂。所述靶向药物选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂,所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药物,所述免疫检查点阻断剂选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体;所述表观遗传学药物例如组蛋白去乙酰化酶抑制剂,所述靶向PI3K/Akt/mTOR信号通路的抑制剂例如Tricibine,所述酪氨酸激酶抑制剂例如舒尼替尼,所述免疫抑制剂例如环磷酰胺,所述蛋白酶体抑制剂例如硼替佐米,所述免疫抑制剂例如沙利度胺、泊马度胺,所述细胞毒药物例如吉西他滨、替莫唑胺,所述细胞周期非特异性药物例如米托蒽醌。
该试剂盒可以包含涉及治疗有效量的包含如上所述的突变体或者蛋白和治疗有效量的治疗剂施用(例如,剂量信息、给药时间间隔信息)的说明书。所述治疗剂选自病毒载体药物、抗体、能降低血液IgG水平的多肽药物;所述病毒载体药物优选溶瘤病毒、基因治疗病毒。
可利用成熟完善的表达系统来制造病毒载体药物。一些方法示例包括利用哺乳动物细胞表达系统来产生病毒颗粒,例如使用HEK293细胞生产腺病毒类病毒载体药物(Freedman Joshua D,Duffy Margaret R,Lei-Rossmann Janet等,An Oncolytic VirusExpressing a T-cell Engager Simultaneously Targets Cancer andImmunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865)。
药物载剂可以是液体,并且药物组合物可以为溶液形式。液体载剂用于制备溶液、悬浮液、乳液、糖浆、酏剂和加压组合物。活性成分可以溶解或悬浮在药学上可接受的液体载剂中,例如水、有机溶剂、二者的混合物或药学上可接受的油或脂肪。
用于肠胃外施用的药物组合物是无菌的、基本上等渗的、无热原的,并根据FDA或类似机构的GMP来制备。病毒载体药物可以作为该物质的溶液或悬浮液的可注射剂型施用,其中,该物质处在生理上可接受的稀释剂和药物载剂(可以是无菌液体,例如水、油、盐水、甘油或乙醇)中。另外,组合物中可存在辅助物质,例如润湿剂或乳化剂、表面活性剂和pH缓冲物质等。药物组合物的其他组分有石油、动物、植物或合成来源的组分,例如花生油、大豆油和矿物油。通常,例如丙二醇或聚乙二醇等二醇是优选的液体载剂,对于可注射溶液尤其如此。病毒载体药物可以以积存注射剂或植入制剂的形式施用,这些形式能够被配制成允许活性成分持续释放。通常,将组合物制备成可注射物,即液体溶液或悬浮液;也可以制备成适合于在注射前溶解或悬浮在液体载质中的固体形式。
除非另外定义,否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料,但现在描述优选方法、装置和材料。
术语“核苷酸”或者“多核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制,否则所述术语涵盖含有天然核苷酸的已知类似物的核酸,所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制,否则所述术语也意指寡核苷酸类似物,其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定,否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言,可通过产生其中一个或一个以上所选(或所有)密码子的第3位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代(Batzer等,Nucleic Acid Res.19:5081(1991);Ohtsuka等,J.Biol.Chem.260:2605-2608(1985);和Cassol等,(1992);Rossolini等,Mol Cell.Probes8:91-98(1994))。
术语“多肽”和“蛋白”在本文中互换使用以意指氨基酸残基的聚合物。即,针对多肽的描述同样适用于描述肽和描述蛋白,且反之亦然。所述术语适用于天然产生氨基酸聚合物以及其中一个或一个以上氨基酸残基为非天然编码氨基酸的氨基酸聚合物。如本文中所使用,所述术语涵盖任何长度的氨基酸链,其包括全长蛋白(即抗原),其中氨基酸残基经由共价肽键连接。
术语“宿主细胞”意指包含本发明核苷酸的细胞,而不管使用何种方法进行插入以产生重组宿主细胞,例如直接摄取、转导、配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。宿主细胞可为原核细胞或真核细胞。
术语“转化”意指将异源性DNA序列引入到宿主细胞或有机体的方法。
术语“表达”意指内源性基因或转基因在细胞中的转录和/或翻译。
本发明的积极进步效果在于:本发明提供一种免疫球蛋白降解酶突变体,其活性和/或热稳定性高于野生型IdeE,活性也高于IdeS和IdeZ(在切割人IgG上比IdeS和IdeZ更有效,活性接近IdeS的2倍,超过IdeZ的4倍)。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1.突变体文库的设计和表达
设计构建野生型IdeE蛋白序列突变文库,经过筛选从中获得40个突变体菌株。
通过密码子优化后合成编码野生型IdeE蛋白序列(SEQ ID NO:2)的多核苷酸序列,并加入N端信号肽序列和C末端6×组氨酸标签,序列合成后插入到pET32a表达载体中,测序正确后获得用于表达野生型IdeE的重组质粒。基于野生型IdeE的表达质粒,设计突变文库所需兼并引物,对原始野生型序列进行扩增,扩增后序列插入到载体上获得突变文库重组质粒。将野生型和突变文库重组质粒电转化到大肠杆菌BL21 Star(DE3)中,并接种在含100ug/ml氨苄青霉素的LB琼脂糖平板上。37℃培养过夜至菌落长出。挑取单个菌落,接于200ul含100ug/ml氨苄青霉素的LB培养基中,37℃、250rpm培养过夜。过夜培养物接于1ml含100ug/ml氨苄青霉素的LB培养基中,37℃培养4h后加入0.1mM IPTG,30℃继续培养过夜。过夜培养物通过离心收集上清。使用SDS-PAGE来评价突变体表达上清中突变体蛋白的浓度。
实施例2.突变体对人IgG1切割活性的评估
为了评价各突变体对人IgG1的切割活性,建立了基于ELSIA的活性测定方法。测定原理是用人IgG1特异性抗原包被酶标板,然后将含有相当浓度突变体蛋白的上清样品与人IgG1在孔中一起温育。使用针对抗体Fc部分具有特异性的人IgG1检测抗体来测量与孔结合的完整或未被完全切割的人IgG1的量。在孔中给定的上清中突变体蛋白浓度相同的情况下,突变体蛋白切割人IgG1活性越高,则越少的完整人IgG1抗体与孔结合,从而得到越低的信号。以不同浓度IgG1与对应检测信号之间的关系可制作IgG1标准曲线,根据标准曲线计算完整或未被完全切割的IgG1的量,进而计算被完全切割的IgG1的量。以被完全切割的IgG1占初始IgG1的比例来评价突变体活性的高低。
为了将实施例1中收获的上清中突变体蛋白浓度处于相当的水平,以相同的上样量进行SDS-PAGE检测,使用Quantity One对电泳图谱中目标蛋白条带的光密度值进行分析,在上样量相同的情况下,图谱中目标蛋白条带的光密度值越高,浓度越高。以IdeE上清作为对照,将其他突变体上清进行浓缩或稀释,使得突变体蛋白条带的光密度值都和IdeE对照的光密度值基本一致。
将上清中蛋白浓度调整至相当的水平后按照如下方法进行ELISA检测:将酶标板用2ug/ml人IgG1(曲妥珠单抗)特异性抗原(货号QRE-104,瑞安生物)在2~8℃包被过夜,然后用PBST(PBS+0.05%吐温20)洗涤,洗涤后的酶标板用2%BSA(PBS配制)在37℃封闭2h,封闭后PBST洗涤。
标准曲线制作:用200ng/ml曲妥珠单抗用反应缓冲液(10mM PB,10mM NaCl,pH6.5)以1:2比例进行梯度稀释直至3.125ng/ml,将100ul不同浓度的曲妥珠单抗加入酶标板的孔中,用于底物(曲妥珠单抗)标准曲线制作。
切割反应:将蛋白浓度调整后的上清用反应缓冲液(10mM PB,10mM NaCl,pH6.5)稀释5倍,将50ul 100ng/ml曲妥珠单抗和50ul稀释后的上清加入酶标板的孔中。
将酶标板放在37℃振荡温育1h,用PBST洗涤后再将40ng/ml Goat anti-HumanIgG Fc Cross-Adsorbed Secondary Antibody-HRP(货号31413,Thermo)加入孔板中,在37℃振荡温育1h,用PBST洗涤后加入TMB作为HRP的显色底物温育15min,用2N H2SO4终止。用酶标仪检测在450nm处的吸光度。根据底物标准曲线计算不同测试孔中的完整或未被完全切割的曲妥珠单抗浓度,进而计算被完全切割的曲妥珠单抗占初始曲妥珠单抗的比例,以此来评价不同突变体的活性。
各突变体活性相对于野生型IdeE活性的倍数关系如表1所示。实施例1中筛选获得的40个突变体都具有高于野生型IdeE的活性,其中15个突变体的活性为野生型IdeE的2倍或2倍以上。
表1.通过ELISA测得的突变体相对于野生型IdeE活性的倍数关系
实施例3.突变体热稳定性的评估
从表1中显示的15个活性是野生型IdeE的2倍或2倍以上的突变体中选取12个突变体来检测其热稳定性。检测方法活性检测方法如下:
将野生型或各突变体上清分两份,分别放置4℃和50℃条件下保温1h,保温后按照实施例2中的ELISA方法检测野生型或各突变体活性,用50℃保温的活性/4℃保温后的活性计算野生型或各突变体在50℃条件下保温1h的剩余活性百分比(%),并以此来比较野生型和各突变体的热稳定性。
突变体活性相对于野生型IdeE的热稳定性的倍数关系如表2所示。表2显示12个突变体都具有高于野生型的热稳定性,其中有7个突变体热稳定性是野生型的3倍以上。
表2.突变体相对于野生型IdeE热稳定性的倍数关系
实施例4.单点突变体切割人IgG1活性的比较
检测如表1和表2中显示的T8D、T8W、T24A、A59L、A59V、E97D和R280H这7个“活性是野生型IdeE的2倍以上,热稳定性是野生型IdeE的3倍以上”的单点突变体切割人IgG1的活性。
1、突变体的表达和纯化
从实施例1中上述5个单点突变体的转化的平板上各挑1单菌落,接于3ml含100ug/ml氨苄青霉素的LB培养基中,37℃、250rpm培养过夜。过夜培养物接于50ml含100ug/ml氨苄青霉素的LB培养基中,37℃培养至OD600达到0.4~0.6,加入0.1mM IPTG,30℃继续培养过夜。过夜培养物通过离心收集上清。上清再用IDA-Ni琼脂糖磁珠纯化,纯化洗脱的蛋白用超滤离心管再换液至PBS缓冲体系中。使用SDS-PAGE来评价纯化后的突变体蛋白纯度。检测OD280,根据消光系数计算纯化后的突变体蛋白浓度。
2、突变体切割人IgG1活性的比较
通过SDS-PAGE上显现有每种突变体的不同浓度对人IgG1所产生的切割产物来进一步评价不同突变体相对于野生型IdeE的切割人IgG1活性的高低。将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL和0.001mg/mL。分别取50ul不同浓度的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图1展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。图2展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。7个单点突变体在0.001mg/ml浓度下切割IgG1的效果都不差于0.002mg/ml野生型IdeE,说明7个单点突变体的切割人IgG1的活性不低于野生型IdeE的2倍。
实施例5.N端截短截短突变体切割人IgG1活性和热稳定性的比较
在野生型IdeE基础上分别删除N端前15个(D1-V15)、前16个(D1-P16)、前17个(D1-H17)、前18个(D1-Q18)和前19个氨基酸(D1-I19),构建5个N端截短突变体(见表3)。
表3.截短突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
WT_del15 | 删除SEQ ID NO:2前15个氨基酸 | 18 |
WT_del16 | 删除SEQ ID NO:2前16个氨基酸 | 19 |
WT_del17 | 删除SEQ ID NO:2前17个氨基酸 | 20 |
WT_del18 | 删除SEQ ID NO:2前18个氨基酸 | 21 |
WT_del19 | 删除SEQ ID NO:2前19个氨基酸 | 22 |
1、突变体的表达和纯化
按照实施例1的方法合成表3中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图3展示了5个截短突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。5个截短突变体切割活性都与野生型IdeE没有明显差别。
3、突变体热稳定性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.1mg/ml,50℃条件下保温1h,保温后再稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图4展示了50℃条件下保温1h后5个截短突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。50℃热处理后5个截短突变体的残余活性明显高于野生型,说明5个截短突变体热稳定性相对于野生型都有明显提高。
实施例6.C端截短截短突变体切割人IgG1活性的比较
在野生型IdeE基础上分别删除C端最后5个(W311-S315)和最后10个氨基酸(S306-S315),构建2个C端截短突变体(见表4)。
表4.截短突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
WT_delC5 | 删除SEQ ID NO:2后5个氨基酸 | 23 |
WT_delC10 | 删除SEQ ID NO:2后10个氨基酸 | 24 |
1、突变体的表达和纯化
按照实施例1的方法合成表4中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图5展示了2个C端截短突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。2个截短突变体切割活性都比野生型IdeE高出2倍以上。
实施例7.组合突变体切割人IgG1活性和热稳定性的比较
在T24A、A59L、A59V、E97D和R280H 5个单点突变体的基础上分别删除前18个(D1-Q18)氨基酸,构建5个组合突变体(见表5)。
表5组合突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
T24A_del18 | 删除SEQ ID NO:9前18个氨基酸 | 25 |
A59L_del18 | 删除SEQ ID NO:13前18个氨基酸 | 26 |
A59V_del18 | 删除SEQ ID NO:14前18个氨基酸 | 27 |
E97D_del18 | 删除SEQ ID NO:15前18个氨基酸 | 28 |
R280H_del18 | 删除SEQ ID NO:16前18个氨基酸 | 29 |
1、突变体的表达和纯化
按照实施例1的方法合成表5中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图6展示了5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。比较图6和图2的切割效果,5个截短突变体和单点组合突变体切割活性没有明显差别,说明组合突变体切割人IgG1的活性同样不低于野生型IdeE的2倍。
3、突变体热稳定性的比较
将纯化后的突变体分别稀释至0.1mg/ml,50℃条件下保温1h,保温后再稀释至0.001mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图7展示了50℃条件下保温1h后5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。比较图8和图7的切割效果,5个组合突变体50℃热处理后活性只是略有下降,说明5个组合突变体相对于野生型热稳定性也都有明显提高。
实施例8.E97D_del18突变体与IdeS和IdeZ活性比较
将实施例7中纯化的E97D_del18突变体依次稀释至20μg/mL,10μg/mL,5μg/mL,2.5μg/mL和1.25μg/ml。将IdeS(货号:A0-FRI-020,Genovis)按照其标识分别稀释至2U/μl,1U/μl,0.5U/μl,0.25U/μl和0.125U/μl。将IdeZ(货号:A0-FRZ-020,Genovis)分别稀释至0.4U/μl,0.2U/μl,0.1U/μl,0.05U/μl和0.025U/μl。分别取50μl不同浓度的突变体、IdeS或IdeZ加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图8展示了不同浓度E97D_del18突变体和IdeS切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeS酶浓度介于7号和8号泳道E97D_del18突变体酶浓度之间,由此递推3号泳道IdeS酶浓度介于9号和10号泳道E97D_del18突变体酶浓度,而3号泳道的IgG1的酶切效果介于10号和11号泳道,由此可以推断E97D_del18突变体切割人IgG1的活性接近IdeS的2倍。
图9展示了不同浓度E97D_del18突变体和IdeZ切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeZ酶浓度高于7号泳道E97D_del18突变体酶浓度,由此递推3号泳道IdeZ酶浓度高于9号泳道E97D_del18突变体酶浓度,即高于11号泳道E97D_del18突变体酶浓度的4倍,而3号泳道的IgG1的酶切效果接近11号泳道,由此可以推断E97D_del18突变体切割人IgG1的活性高于IdeZ的4倍。
实施例9.E97D_del18突变体切割人IgG1活性的体外检测
通过检测加入E97D_del18突变体与人IVIg处理过的小鼠血清或血浆中完整或单切割IVIg的量来评价E97D_del18突变体对人IgG1的体外切割活性。按照表6配制不同组别的小鼠血清或血浆酶切体系。
表6.小鼠血清或血浆酶切体系
碘乙酸处理组中碘乙酸的作用是抑制IgG降解酶的活性。
将体系置于37℃反应30min。取20μl样品与同等体积的2×SDS非还原上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图10展示了E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物电泳图。结果显示E97D_del18在小鼠血清和血浆中都可以有效切割人IVIg。
通过检测加入E97D_del18突变体处理过的小鼠或人血清来评价E97D_del18突变体是否具有对人IgG1的体外切割活性。按照表7配制不同组别的小鼠或人血清酶切体系。
表7.小鼠或人血清酶切体系
将体系置于37℃反应24h。取20μl样品与同等体积的2×SDS还原性上样缓冲液混合,再用1×SDS还原性上样缓冲液稀释20倍,75℃水浴5min,SDS-PAGE检测切割产物。
图11展示了E97D_del18突变体在小鼠和人血清中产生的切割产物电泳图。结果显示E97D_del18在人血清中切割产生明显可见的25kD的Fc片段,而在小鼠血清中则没有该片段,说明E97D_del18能有效特异性切割人血清中IgG1,而对小鼠血清中的IgG1切割活性很低或无切割活性。
实施例10.E97D_del18突变体切割不同种属的免疫球蛋白
通过检测加入E97D_del18突变体与不同种属动物血清或血浆中完整或单切割IgG的量来评价E97D_del18突变体对不同种属动物血清免疫球蛋白的体外切割活性。按照表8和表9配制不同种属的血清或抗体酶切体系。
表8比格犬血清及不同物种抗体酶切体系
将体系置于37℃反应1小时,酶切产物采用SDS-PAGE检测。
表9不同物种血清酶切体系
图12A-12D展示了E97D_del18突变体在不同物种血清和抗体的效果。结果显示E97D_del18可有效切割犬IgG、兔IgG以及小鼠IgG2a,无法切割小鼠IgG1;E97D_del18可有效切割兔、犬和猴血清IgG,其中兔血清IgG切割效果最好,猪血清IgG切割效果较差,大鼠和小鼠血清IgG则几乎未被切动。
实施例11.人体内针对E97D_del18突变体的预存抗体低
该测定是基于E97D_del18突变体与IdeS之间对于与抗E97D_del18/IdeS抗体结合的竞争。测试酶和人血清的预温育将使得抗E97D_del18/IdeS抗体与E97D_del18突变体与IdeS能够结合。
将E97D_del18突变体与IdeS在孔板上包被过夜,然后用PBST洗涤并在2%BSA封闭液中封闭1小时,用逐步稀释的待测突变体与IdeS和人血清制备混合板,将混合版在室温下振荡温育1小时,PBST洗涤之后加入生物素标记的E97D_del18突变体与IdeS,再加入SA-HRP,用TMB显色并读数。平行比对获得约80个人血样本中E97D_del18和IdeS预存抗体的情况。
结果如表10所示,IdeS在正常人血清中预存抗体的比例高达约90%,而E97D_del18突变体仅为约20%。E97D_del18突变体在体内的预存抗体明显少于IdeS,证明E97D_del18突变体的免疫原性更低,更有利于体内用药。
表10.E97D_del18突变体和IdeS在人血样本中预存抗体比对情况
E97D_del18突变体 | IdeS | |
人血样本总数(例) | 76 | 76 |
预存抗体阳性样本比例(%) | 18.4 | 89.5 |
实施例12.E97D_del18突变体切割人IgG1活性的体内检测
在无菌条件下用人IVIg(静注人免疫球蛋白)腹腔注射2只小鼠(两只小鼠为平行实验,小鼠编号1号和2号),注射剂量为1g/kg。注射人IVIg 24h后,再将IgG降解酶(E97D_del18)以5mg/kg的剂量静脉注射小鼠,2只小鼠都分别在注射E97D_del18后0h、15min、2h、6h和24h采血并收集血清样品。取20μl血清样品与同等体积的2×SDS非还原上样缓冲液混合后,再用1×SDS非还原性上样缓冲液稀释20倍,75℃水浴5min,SDS-PAGE检测。
图13展示了E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物电泳图。结果显示,E97D_del18在小鼠体内切割IVIg效果显著,15min基本已经达到完全酶切。
实施例13.组合突变体切割人IgG1活性的比较
在上述突变体的基础上,进一步构建了6种组合突变体,所述突变的序列如表11所示。
表11.组合突变体
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
E97D_del18_delC5 | 删除SEQ ID NO:28后5个氨基酸 | 30 |
E97D_del18_delC10 | 删除SEQ ID NO:28后10个氨基酸 | 31 |
A59V_del18_delC5 | 删除SEQ ID NO:27后5个氨基酸 | 32 |
A59L_del18_delC5 | 删除SEQ ID NO:26后5个氨基酸 | 33 |
R280H_del18_delC5 | 删除SEQ ID NO:29后5个氨基酸 | 34 |
E97D_A59V_R280H | E97D、A59V、R280H三突变组合 | 35 |
1、突变体的表达和纯化
按照实施例1的方法合成表11中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3)。按照实施例4中的方法制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50ul稀释后的突变体或野生型IdeE加入50ul含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30min。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图14A和14B展示了6个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (18)
1.免疫球蛋白降解酶IdeE的突变体,其特征在于,所述的免疫球蛋白降解酶IdeE包含如序列表中SEQ ID NO:2所示的氨基酸序列或由所述氨基酸序列组成;所述突变选自下组:
(1)对所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体;和/或,
(2)对所述免疫球蛋白降解酶IdeE进行截短,删除其N端的前1个、前2个、前3个、前4个、前5个、前6个、前7个、前8个、前9个,前10个、前11个、前12个、前13个、前14个、前15个、前16个、前17个、前18个或者前19个氨基酸序列;和/或,
(3)对所述免疫球蛋白降解酶IdeE进行截短,删除其C端的最后1个、最后2个、最后3个、最后4个、最后5个、最后6个、最后7个、最后8个、最后9个或者最后10个氨基酸序列;
其中所述的突变体具有高于所述免疫球蛋白降解酶IdeE的活性,和/或具有高于所述免疫球蛋白降解酶IdeE的热稳定性。
2.如权利要求1所述的突变体,其特征在于所述突变选自下组:
(1)对SEQ ID NO:2所示的氨基酸序列的位置8、10、24、59、97或者280进行替换;和/或
(2)删除所述免疫球蛋白降解酶IdeE N端的前15个、前16个、前17个、前18个或者前19个氨基酸;优选删除前18个氨基酸;和/或
(3)删除所述免疫球蛋白降解酶IdeE C端的最后5个或者最后10个氨基酸;优选删除最后5个氨基酸。
3.如权利要求1或2所述的突变体,其特征在于所述替换选自下组:
(1)所述位置8的苏氨酸被替换成半胱氨酸、苯丙氨酸、色氨酸、酪氨酸、天冬氨酸、谷氨酸、丙氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、丝氨酸、缬氨酸、精氨酸和赖氨酸中的任意一种;
(2)所述位置10的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(3)所述位置24的苏氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、天冬酰胺、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(4)所述位置59的丙氨酸被替换成半胱氨酸、天冬氨酸、谷氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;
(5)所述位置97的谷氨酸被替换成丙氨酸、半胱氨酸、天冬氨酸、苯丙氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、精氨酸、丝氨酸、苏氨酸、缬氨酸、色氨酸和酪氨酸中的任意一种;和
(6)所述位置280的精氨酸被替换成丙氨酸、天冬氨酸、谷氨酸、半胱氨酸、丝氨酸、苯丙氨酸、组氨酸、异亮氨酸、赖氨酸、亮氨酸、蛋氨酸、天冬酰胺、脯氨酸、谷氨酰胺、色氨酸、苏氨酸、缬氨酸和酪氨酸中的任意一种。
4.如权利要求3所述的突变体,其特征在于,所述的突变体如序列表中SEQ ID NO:3~35任一个所示。
5.一种包含如权利要求1~4任一项所述的突变体的蛋白。
6.如权利要求5所述的蛋白,其特征在于,所述蛋白在所述突变体的N末端包含信号肽;优选地,所述蛋白在所述突变体的N末端连接有分泌信号序列并在所述分泌序列的N末端连接有甲硫氨酸和/或在在所述突变体C末端连接有组氨酸标签;更优选地,所述蛋白从N末端至C末端由如下组成:甲硫氨酸、分泌信号序列和所述突变体。
7.一种编码如权利要求1~4任一项所述的突变体或者如权利要求5或6所述的蛋白的核苷酸。
8.一种包含如权利要求7所述的核苷酸的表达载体。
9.一种包含如权利要求8所述的表达载体或者表达如权利要求1~4任一项所述的突变体或者如权利要求5或6所述的蛋白的宿主细胞;所述宿主细胞优选为大肠杆菌细胞或者酵母细胞。
10.一种组合物,其包含:
免疫球蛋白降解酶或其突变体或者包含所述免疫球蛋白降解酶或其突变体的蛋白;和
任选的药学上可接受的载体或赋形剂。
11.如权利要求10所述的组合物,其中所述免疫球蛋白降解酶选自IdeE、IdeS和IdeZ。
12.如权利要求10或11所述的组合物,其中所述免疫球蛋白降解酶的突变体为如权利要求1~4任一项所述的突变体,或者所述包含所述免疫球蛋白降解酶或其突变体的蛋白为如权利要求5或6所述的蛋白。
13.如权利要求10-12中任一项所述的组合物,其进一步包含:
抗体或者含有Fc的蛋白。
14.如权利要求13所述的组合物,其中所述抗体的靶点选自下组:细胞表面蛋白、细胞因子、激素、酶、胞内信使、胞间信使和免疫检查点。
15.如权利要求10-14中任一项所述的组合物,其进一步包含:
病毒载体药物,优选地,所述病毒载体药物选自下组:溶瘤病毒、基因治疗病毒和病毒载体疫苗。
16.如权利要求10-15中任一项所述的组合物,其进一步包含:
能降低血液IgG水平的药物,优选的,所述降低血液IgG水平的药物选自下组:FcRn抗体、与FcRn高亲和力的Fc片段变体。
17.一种试剂盒,其包含:
(1)如权利要求1~4任一项所述的突变体或者如权利要求5或6所述的蛋白;和
(2)选自下组的一种或多种:(a)药学上可接受的载体或赋形剂;(b)抗体或者包含Fc的蛋白;和/或
(3)病毒载体药物,所述病毒载体药物选自溶瘤病毒、基因治疗病毒和病毒载体疫苗;和/或
(4)能降低血液IgG水平的药物,所述降低血液IgG水平的药物选自FcRn抗体、与FcRn高亲和力的Fc片段变体。
18.一种试剂盒,其包含:药盒A和药盒B,其特征在于,
所述的药盒A含有如权利要求1~4任一项所述的突变体或者如权利要求5或6所述的蛋白,
所述的药盒B含有选自下组的一种或多种:
(1)药学上可接受的载体或赋形剂;(2)抗体或者包含Fc的蛋白;和/或(3)病毒载体药物;和/或(4)能降低血液IgG水平的药物;
其中,所述病毒载体药物选自溶瘤病毒、基因治疗病毒和病毒载体疫苗;所述降低血液IgG水平的药物选自FcRn抗体、与FcRn高亲和力的Fc片段变体。
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010557830 | 2020-06-18 | ||
CN202010557830X | 2020-06-18 | ||
CN202180013436.2A CN115443288A (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
PCT/CN2021/100844 WO2021254479A1 (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202180013436.2A Division CN115443288A (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118562771A true CN118562771A (zh) | 2024-08-30 |
Family
ID=79268498
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202180013436.2A Pending CN115443288A (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
CN202410654527.XA Pending CN118562771A (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202180013436.2A Pending CN115443288A (zh) | 2020-06-18 | 2021-06-18 | 一种免疫球蛋白降解酶IdeE的突变体 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20230364207A1 (zh) |
EP (1) | EP4169934A4 (zh) |
JP (1) | JP2023532219A (zh) |
CN (2) | CN115443288A (zh) |
WO (1) | WO2021254479A1 (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20240123827A (ko) | 2021-12-16 | 2024-08-14 | 상하이 바오 파마슈티컬스 컴퍼니 리미티드 | 항면역글로불린 분해 효소에 의해 효소 절단된 Fc 변이체 |
CN118382454A (zh) * | 2021-12-22 | 2024-07-23 | 上海宝济药业股份有限公司 | 一种免疫球蛋白降解酶IdeE的突变体的用途 |
CN114875003B (zh) * | 2022-04-06 | 2024-05-24 | 浙江大学 | 一种短链脱氢酶的突变体、编码基因及编码基因获得方法、突变体的应用 |
EP4349365A1 (en) | 2022-10-07 | 2024-04-10 | Hansa Biopharma AB | Co-treatment for gene therapy |
CN118497176A (zh) * | 2024-07-16 | 2024-08-16 | 苏州康聚生物科技有限公司 | 一种免疫球蛋白降解酶 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011506433A (ja) * | 2007-12-13 | 2011-03-03 | インターバック アーベー | 改変された免疫組成物 |
WO2011149419A1 (en) * | 2010-05-26 | 2011-12-01 | Intervacc Ab | Vaccine against streptococcal infections based on recombinant proteins |
GB201502305D0 (en) * | 2015-02-12 | 2015-04-01 | Hansa Medical Ab | Protein |
GB201502306D0 (en) * | 2015-02-12 | 2015-04-01 | Hansa Medical Ab | Protein |
EP3319630A1 (en) * | 2015-07-09 | 2018-05-16 | Intervacc AB | Vaccine against s.suis infection |
US11001819B2 (en) * | 2015-07-16 | 2021-05-11 | Daiichi Sankyo Company, Limited | Endos mutant enzyme |
JP7123801B2 (ja) * | 2016-02-04 | 2022-08-23 | ジェノビス エービー | 新しい連鎖球菌プロテアーゼ |
WO2018093868A1 (en) * | 2016-11-16 | 2018-05-24 | University Of Florida Research Foundation, Inc. | Immunoglobulin proteases, compositions, and uses thereof |
WO2022057942A1 (zh) * | 2020-09-21 | 2022-03-24 | 上海宝济药业有限公司 | 一种药物组合及其应用 |
-
2021
- 2021-06-18 EP EP21825273.2A patent/EP4169934A4/en active Pending
- 2021-06-18 CN CN202180013436.2A patent/CN115443288A/zh active Pending
- 2021-06-18 CN CN202410654527.XA patent/CN118562771A/zh active Pending
- 2021-06-18 JP JP2022577764A patent/JP2023532219A/ja active Pending
- 2021-06-18 US US18/001,876 patent/US20230364207A1/en active Pending
- 2021-06-18 WO PCT/CN2021/100844 patent/WO2021254479A1/zh unknown
Also Published As
Publication number | Publication date |
---|---|
EP4169934A4 (en) | 2024-07-24 |
CN115443288A (zh) | 2022-12-06 |
WO2021254479A1 (zh) | 2021-12-23 |
EP4169934A1 (en) | 2023-04-26 |
US20230364207A1 (en) | 2023-11-16 |
JP2023532219A (ja) | 2023-07-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN118562771A (zh) | 一种免疫球蛋白降解酶IdeE的突变体 | |
CA3055202A1 (en) | Cd19 compositions and methods for immunotherapy | |
US11142580B2 (en) | Oncolytic virus and method | |
AU2018351072B2 (en) | Systems and methods to produce B cells genetically modified to express selected antibodies | |
JP7441840B2 (ja) | 変異したpiggybacトランスポザーゼ | |
WO2021244628A1 (zh) | 一种酶和病毒的药物组合及其应用 | |
Mensah et al. | Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins | |
JP6920786B2 (ja) | 免疫ウイルス療法のためのrnaウイルス | |
WO2023116817A1 (zh) | 一种免疫球蛋白降解酶IdeE的突变体的用途 | |
US12030938B2 (en) | Engineered chimeric fusion protein compositions and methods of use thereof | |
US20210254106A1 (en) | Engineering b lymphocytes by utilizing endogenous activation-induced cytidine deaminase | |
CN113769058B (zh) | 一种药物组合及其应用 | |
US20220307039A1 (en) | Codon-optimized nucleotide sequences encoding an ap-1 transcription factor | |
Pflumm | Development of a novel SARS-CoV-2 Vaccination Strategy with a monomeric Spike Designer Antigen to prime efficient Antibody Responses in aged Mice | |
Azar et al. | TG6050, an oncolytic vaccinia virus encoding interleukin-12 and anti-CTLA-4 antibody, favors tumor regression via profound immune remodeling of the tumor microenvironment | |
CN118359730A (zh) | 包含细胞因子的靶向型融合蛋白 | |
EA046539B1 (ru) | Системы и способы получения в-клеток, генетически модифицированных, для экспрессии выбранных антител | |
NZ791667A (en) | Adenovirus armed with bispecific T-cell activator |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination |