CN1381582A - Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage - Google Patents
Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage Download PDFInfo
- Publication number
- CN1381582A CN1381582A CN 01105972 CN01105972A CN1381582A CN 1381582 A CN1381582 A CN 1381582A CN 01105972 CN01105972 CN 01105972 CN 01105972 A CN01105972 A CN 01105972A CN 1381582 A CN1381582 A CN 1381582A
- Authority
- CN
- China
- Prior art keywords
- adenovirus
- csf
- stimulating factor
- recombinant adenovirus
- district
- 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.)
- Granted
Links
- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 139
- 238000000034 method Methods 0.000 title claims abstract description 37
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 title claims description 51
- 230000008569 process Effects 0.000 title abstract description 5
- 241000282414 Homo sapiens Species 0.000 claims abstract description 21
- 210000004027 cell Anatomy 0.000 claims description 74
- 230000014509 gene expression Effects 0.000 claims description 42
- 108090000623 proteins and genes Proteins 0.000 claims description 34
- 241000700605 Viruses Species 0.000 claims description 28
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 21
- 101150013553 CD40 gene Proteins 0.000 claims description 20
- 208000015181 infectious disease Diseases 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 10
- 239000013601 cosmid vector Substances 0.000 claims description 9
- 241000701022 Cytomegalovirus Species 0.000 claims description 7
- 210000004907 gland Anatomy 0.000 claims description 7
- 102100032912 CD44 antigen Human genes 0.000 claims description 6
- 102100035793 CD83 antigen Human genes 0.000 claims description 6
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims description 6
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 claims description 6
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 claims description 6
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 claims description 6
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 claims description 6
- 238000012217 deletion Methods 0.000 claims description 6
- 230000037430 deletion Effects 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 230000008521 reorganization Effects 0.000 claims description 6
- 230000008488 polyadenylation Effects 0.000 claims description 5
- 241000701822 Bovine papillomavirus Species 0.000 claims description 4
- -1 CD86 Proteins 0.000 claims description 4
- 239000003981 vehicle Substances 0.000 claims description 4
- 239000001506 calcium phosphate Substances 0.000 claims description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 3
- 235000011010 calcium phosphates Nutrition 0.000 claims description 3
- 238000000975 co-precipitation Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 11
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 210000003714 granulocyte Anatomy 0.000 abstract description 6
- 210000002540 macrophage Anatomy 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000009169 immunotherapy Methods 0.000 abstract description 2
- 230000004936 stimulating effect Effects 0.000 abstract description 2
- 239000002131 composite material Substances 0.000 abstract 1
- 230000001524 infective effect Effects 0.000 abstract 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 64
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 64
- 108020004414 DNA Proteins 0.000 description 39
- 102000046157 human CSF2 Human genes 0.000 description 29
- 238000002360 preparation method Methods 0.000 description 18
- 239000013598 vector Substances 0.000 description 16
- 230000003612 virological effect Effects 0.000 description 16
- 230000006801 homologous recombination Effects 0.000 description 14
- 238000002744 homologous recombination Methods 0.000 description 14
- 239000012634 fragment Substances 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 210000004443 dendritic cell Anatomy 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- 238000012856 packing Methods 0.000 description 10
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 230000003321 amplification Effects 0.000 description 8
- 238000001415 gene therapy Methods 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108020005202 Viral DNA Proteins 0.000 description 6
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 6
- 230000036039 immunity Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 239000013605 shuttle vector Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 239000002516 radical scavenger Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 210000001163 endosome Anatomy 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 210000004493 neutrocyte Anatomy 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 210000002845 virion Anatomy 0.000 description 4
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 3
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 230000003263 anti-adenoviral effect Effects 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000002574 poison Substances 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 238000002525 ultrasonication Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- CXURGFRDGROIKG-UHFFFAOYSA-N 3,3-bis(chloromethyl)oxetane Chemical compound ClCC1(CCl)COC1 CXURGFRDGROIKG-UHFFFAOYSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 208000032467 Aplastic anaemia Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010322 bone marrow transplantation Methods 0.000 description 2
- 102220369445 c.668T>C Human genes 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 238000012637 gene transfection Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 230000011132 hemopoiesis Effects 0.000 description 2
- 230000002607 hemopoietic effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 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 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010051113 Arterial restenosis Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 208000027219 Deficiency disease Diseases 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 208000003556 Dry Eye Syndromes Diseases 0.000 description 1
- 206010013774 Dry eye Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 102100031939 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 101710145505 Fiber protein Proteins 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 206010018687 Granulocytopenia Diseases 0.000 description 1
- 241000606790 Haemophilus Species 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- 101001086862 Homo sapiens Pulmonary surfactant-associated protein B Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 208000009319 Keratoconjunctivitis Sicca Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 1
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 102100032617 Pulmonary surfactant-associated protein B Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 241001068295 Replication defective viruses Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 206010042566 Superinfection Diseases 0.000 description 1
- 108010087302 Viral Structural Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000013228 adenopathy Diseases 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 208000006682 alpha 1-Antitrypsin Deficiency Diseases 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 201000003146 cystitis Diseases 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000000222 eosinocyte Anatomy 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 210000002347 langhans' cell Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000000088 lip Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000004777 protein coat Anatomy 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 239000003352 sequestering agent Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009747 swallowing Effects 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Images
Abstract
A recombinant adenovirus for effectively expressing human granulocyte macrophage clony stimulating factor with new target orientation, its preparing process and usage, and the composite medicine containing it are disclosed. It can be used for immunotherapy of tumor and infective disease with low cost and high effect.
Description
The present invention relates to field of gene.More specifically, the present invention relates to a kind of new recombinant adenovirus target, the high expression level human granulocyte macrophage colony stimulating factor, the preparation method of this recombinant adenovirus and purposes, and the pharmaceutical composition that contains this recombinant adenovirus.
(Colony stimulating factor is a class hemopoieticgrowth factor CSF) to G CFS, can stimulate the marrow pluripotential hemopoietic stem cell to respectively being progenitor cell colony differentiation, and make thesocytes such as its bud into mature granulocyte, scavenger cell.CSF comprises multicolony stimulating factor (multipotent CSF), granulocyte-macrophage colony stimutaing factor (Granulocyte-macrophage colony-stimulating factor abbreviates " GM-CSF " as), granulocyte colony-stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) etc.GM-CSF is external, all can promote proliferation and differentiation (Metcalf D.Science, 1985 of granulocyte, scavenger cell and dendritic cell in the body; 229:16-22).After giving GM-CSF in vivo, the white corpuscle of observing persistence raises.In addition, except acting on the not hematopoietic cell of differentiation and maturation, GM-CSF also has important regulatory role to the immunocyte of differentiation and maturation, as suppress neutrophil leucocyte and move, strengthen the neutrophil leucocyte chemotactic, engulf and strengthen monokaryon-scavenger cell antitumor and antibody-dependent cytotoxicity effect (Gasson JC et al.Blood, 1991 such as (ADCC); 77:1131).What especially be subjected in recent years extensively paying attention to is that discovery GM-CSF can strengthen the angtigen presentation function of scavenger cell and dendritic cell etc., shows that it also has broad clinical application prospect in antitumor and anti-infectious immunity treatment.Experimentation on animals shows that the tumour cell of GM-CSF genetic modification can be induced for a long time in vivo, effective specificity antineoplastic immunity rejection, have significant therapeutic effect (Dranoff G.etal, Proc Natl Acad Sci USA, 1993,90:3539).The tumour cell of GM-CSF genetic modification is as a kind of novel and effective tumour knurl seedling at present, be used for clinical trial, late tumor patient (Roth J and Cristiano R.J Natl Cancer Inst.1997 such as treatment melanoma, kidney and prostate cancer; 89:21-39).
Since the cDNA sequence of successfully being cloned into human GM-CSF (Wong G et al.Science, 1985; 228:810.Burgess A et al.J Biol Chem, 1987; 252:1991), GM-CSF is as first Hemopoietic factor (Vlich TR et al.Blood, 1990 that utilize recombinant DNA technology to produce; 75:846), shown the clinical value that it is huge.Using rHuGM-CSF in the body stimulates leukocytic increasing, and mainly is neutrophil leucocyte, eosinophilic granulocyte and monocytic increasing, and lymphocyte only has slightly once in a while and increases.Clinically, macrophage colony stimulating factor of recombinant human granulocyte is widely used in cancer radiation chemotherapy (Silver GM et al.Surgery, 1989; 106:452-455, Salgaller ML et al.J Surg Oncol 1998; 68:122-138), myelodysplastisches syndromes (Vadhan-Raj S et al.Blood, 1989; 74:1491-1498), AIDS (Baldwin GC et al.Blood, 1989; 74:1673-1667), bone marrow transplantation (NemunaitisJ et al.Blood, 1988; Treatment of diseases or assisting therapy such as 72:834-836).In addition, GM-CSF also has comparatively significant curative effect to chronic and heredity neutrophil leucocyte deficiency disease, parasitic infection, aplastic anemia etc.
The human GM-CSF that gene recombination technology is produced now formally is applied to clinical, the patient of the acute and chronic hemopoietic hypofunction of marrow that the treatment a variety of causes causes, and receive significant curative effect.Its main clinical indication comprises: (Biotherapy 1998,10:299 in hematopoiesis recovery of the granulocytopenia that chemotherapy and radiation causes, myelodysplastic syndrome (MDS), aplastic anemia, AIDS and promotion bone marrow transplantation patient or the like; Int J Hematol.1996,65:17; Med Oncol.1996,13:155; Cancer 1996,77:1149).
People such as Tarr have carried out human trial in 1996, the HBV vaccine (injection once) of will recombinating in this experiment share with rhGM-CSF (injection once), both are injected to same position can activate partial antigen presenting cell (as Langhans' cells) to wish GM-CSF, thereby is beneficial to antigenic picked-up processing and offers.Found that have only an example the anti-HBV antibody response of low titre protectiveness to occur 27 routine volunteers of control group, and the experimental group philtrum of subcutaneous injection GM-CSF has 5 examples to produce the high titre antibody responses of protectiveness in 27 examples.Accept in the experimental group of intramuscular injection GM-CSF, and the high titre antibody response of 27 routine patient's 6 example generation protectiveness (Tarr PE, et al.Vaccine 1996,14:1199-204).This description of test, GM-CSF can effectively strengthen the immune effect of knurl seedling as a kind of effective immunological adjuvant.
Yet,, still have some problems GM-CSF being directly used in when clinical.For example the transformation period is shorter in the body of GM-CSF, thereby needs every day during treatment and inject one or many, and it is higher to add the cost of GM-CSF preparation own, so cause medical expense very expensive and therapeutic efficiency is not high yet.In addition,, need near same position, inject repeatedly, make patient very painful in topical application GM-CSF (as treatment during tumour).Therefore, it is low that this area presses for a kind of cost, the method for using GM-CSF easy to use and relevant preparation.Traditional GM-CSF gene therapy has advantages such as with low cost, easy to use, but often gene transfection is high and target is poor.
Purpose of the present invention just provides a kind of method of using GM-CSF clinically and related preparations of novelty, thereby overcome above-mentioned shortcoming of the prior art, make the clinical GM-CSF of using not only with low cost, easy to use, and efficient is higher, specific cells colony can the target sexuality be dyed in the inside and outside.
In a first aspect of the present invention, a kind of recombinant adenovirus of expressing human rHuGM-CSF of target is provided, the genomic deletion E1 district of this adenovirus and E3 district, and inserted the human granulocyte macrophage colony stimulating factor expression cassette in the E1 zone position, this expression cassette comprises successively: promoter sequence, human granulocyte macrophage colony stimulating factor encoding sequence and polyadenylation signal sequence;
And, in the adenovirus surface bonding bi-specific antibody being arranged, this antibody is both specifically at the binding site on adenovirus surface, again specifically at the acceptor molecule of cell surface.
Preferably, described adenovirus is an Ad5 type adenovirus; And described acceptor molecule is selected from down group: CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule.
In another preference, contained promoter sequence comprises the eukaryotic expression promotor that is selected from down group in the human granulocyte macrophage colony stimulating factor expression cassette: cytomegalovirus promoter, bovine papilloma virus promotor.In another preference, the human granulocyte macrophage colony stimulating factor of two copies is arranged in expression cassette.
In a second aspect of the present invention, a kind of pharmaceutical composition is provided, it comprises above-mentioned target and the recombinant adenovirus expressing human rHuGM-CSF and the pharmaceutically acceptable carrier or the vehicle of significant quantity.
In a third aspect of the present invention, a kind of method for preparing recombinant adenovirus target, the expressing human rHuGM-CSF is provided, this method comprises:
(a) provide adenovirus DNA-TP mixture,, and be combined with terminal peptide TP at the two ends of adenovirus genomic dna in this adenovirus DNA genomic deletion E1 district and E3 district;
(b) provide gland-containing virus genomic cosmid vector, adenoviral gene group in this cosmid vector has lacked E1 district and E3 district, and inserted the human granulocyte macrophage colony stimulating factor expression cassette in the E1 zone position, wherein this expression cassette comprises successively: promoter sequence, human granulocyte macrophage colony stimulating factor encoding sequence and polyadenylation signal sequence;
(c) the cosmid vector cotransfection in the adenovirus DNA in the step (a)-TP mixture and the step (b) is expressed the host cell of E1 district gene;
(d) screening positive clone;
(e) separation and purification recombinant adenovirus from positive colony.
(f) adenovirus in the step (e) is combined with bi-specific antibody, this antibody is both specifically at the binding site on adenovirus surface, again specifically at the acceptor molecule of cell surface, thereby obtain recombinant adenovirus target, the expressing human rHuGM-CSF.
Preferably, the cotransfection method is a calcium phosphate DNA co-precipitation infection protocol in step (c), this host cell is 293 cells, and contained promoter sequence is selected from down group in the human granulocyte macrophage colony stimulating factor expression cassette: cytomegalovirus promoter, bovine papilloma virus promotor; Described acceptor molecule is selected from down group: CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule; The human granulocyte macrophage colony stimulating factor that two copies are arranged in the expression cassette.
In still another aspect of the invention, provide a kind of method for preparing the recombinant adenovirus of expressing human rHuGM-CSF, this method comprises:
(a) provide adenovirus DNA-TP mixture,, and be combined with terminal peptide TP at the two ends of adenovirus genomic dna in this adenovirus DNA genomic deletion E1 district and E3 district;
(b) provide gland-containing virus genomic cosmid vector, adenoviral gene group in this cosmid vector has lacked E1 district and E3 district, and inserted the human granulocyte macrophage colony stimulating factor expression cassette in the E1 zone position, wherein this expression cassette comprises successively: promoter sequence, human granulocyte macrophage colony stimulating factor encoding sequence and polyadenylation signal sequence;
(c) the cosmid vector cotransfection in the adenovirus DNA in the step (a)-TP mixture and the step (b) is expressed the host cell of E1 district gene;
(d) screening positive clone;
(e) separation and purification recombinant adenovirus from positive colony.
In the accompanying drawings,
Fig. 1. human GM-CSF cDNA sequence
Fig. 2. human GM-CSF bicistronic mRNA fusion gene and coding protein sequence thereof
Fig. 3 .pShuttle-CMV shuttle vectors physical map
The physical map of Fig. 4 .pAdEasy-1 adenovirus carrier
Fig. 5. homologous recombination produces the recombinant adenovirus synoptic diagram in the bacterium
In the present invention, term " granulocyte macrophage colony stimulating factor coded sequence (GM-CSF) " Refer to naturally occurring or artificial synthetic GM-CSF coded sequence, this coded sequence native form of can encoding GM-CSF, the GM-CSF of the various variant forms of also can encoding (for example the conservative variant of GM-CSF, Allelic variant, natural mutation, induced mutation body, or its active fragment), as long as this variant form GM-CSF has the biologically active of GM-CSF.
In the present invention, term " granulocyte macrophage colony stimulating factor expression cassette " refers to contain GM-CSF Coded sequence and the module of expressing required element are expressed required assembly and are comprised promoter and poly-gland The nucleotide burst. In addition, the granulocyte macrophage colony stimulating factor expression cassette can also contain or Do not contain other sequences, comprising but be not limited to: enhancer, secretion signal peptide sequence etc. Better Ground contains the GM-CSF of two copies or multicopy at the granulocyte macrophage colony stimulating factor expression cassette Coded sequence. A kind of methods that realize that two copies are expressed are to utilize the IRES sequence that the cDNA of GM-CSF is advanced The row fused in tandem, thus realize two copy expression.
In the present invention, term " cell surface receptor molecule " refers at mammalian cell (dendron especially Shape cell, B cell, monocyte or other cells relevant with immunity) cell surface on, can be used as The molecule of antibody combining site. Representational example comprises various CD molecules, for example CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule etc. CD40 be a kind of mainly be expressed in BMDC, The membrane molecule of the cell surfaces such as B cell, monocyte, lower about its corresponding ligand molecular CD40L, The mediated cell activation signals.
In the present invention, go for the startup of human granulocyte macrophage colony stimulus factor expression cassette Son can be any common promoter, and it can be constitutive promoter or inducible promoter. Preferably, this promoter is the strong promoter of composing type, for example cytomegalovirus promoter, cow teats knurl Other is applicable to the promoter of eukaryotic expression viral promotors etc.
In the present invention, available recombined adhenovirus can be the adenovirus of any serotype, Good ground in order to prevent from producing in preparation process parent's replication type adenovirus, should adopt E1 district deletion form gland Virus. More preferably, the adenovirus of using E1 district and E3 district all to lack. In addition, should employing have studied Comparatively thorough adenovirus type, for example Ad2 and Ad5 type adenovirus.
Describe in detail below of the present invention can be used for clinical replication defect type human GM-CSF recombined adhenovirus and Preparation process and the method for relevant pharmaceutical composition (preparation).
1, the viral biology of adenovirus and molecular biological characteristic
Adenovirus is a kind of nonencapsulated dna virus, and virion is regular dodecahedron, and diameter is 80-90nm. Adenovirus major has three kinds of structural proteins: six adjacent bodies, the surface of 20 bodies of formation; Penton, Be arranged in 12 drift angles of protein coat; Fibrin, a fiber initiation on each penton.
The adenoviral gene group is the linear double-stranded DNA of double helix, 36000 base-pairs (bp) is arranged approximately, at two ends Be combined with 55kDa terminal peptide (Terminal peptide, TP). Traditionally viral genome is divided 100 Individual gene map unit (mu), the suitable 360bp of 1mu. Respectively there is the end of an about 100bp at the genome two ends Be inverted inverted repeat dna sequence dna (ITR). Be loaded with viral packaging signal at the genome left end. Gene element is Early transcription district (being divided into E1-E4) and late transcription district (being divided into L1-L5), each transcriptional domain is lived in virus All play an important role in cycle. In addition, also have several little mesozones and late region.
Adenovirus hominis has nearly 50 kinds of serotypes, and wherein Ad2 and Ad5 genome structure and gene expression grinds Study carefully comparatively extensively, now determined to make up the complete nucleotide sequence of this amphitypy adenovirus at present Adenovirus vector comes from this two kinds of serotypes mostly.
E1 district in the adenoviral gene group namely is activated in viral genome one enters host cell nuclear, Thereby the coding initiation factor regulate the early ambulant of virus, so the E1 district is essential by virus replication.
The albumen of E2 district coding is relevant with copying of viral DNA, comprises that the viral DNA end is in conjunction with albumen (TP), DBP (DBP) and archaeal dna polymerase, DBP is relevant with transcriptional control.
The polypeptide of E3 district coding is relevant with the immune response of virus escape host anti-virus, such as, the E3 coding A 19kDa glycoprotein can suppress I class major histocompatibility antigen (MHC) and offer the infection cell table Face makes cytotoxic T lymphocyte (CTL) can not effectively identify and kill and wound infection cell; In addition, E3 compiles 10.4kDa, 24.5kDa, the 14.7kDa albumen of code can suppress the cell of tumor necrosis factor α (TNF-α) Toxic action. E3 district disappearance has no significant effect the infectivity of adenovirus.
E4 district coded product participates in viral dna replication, district's gene expression in evening, virus protein is synthetic and the host The activities such as the synthetic termination of albumen. Main late gene product is virus structural protein, and encoded by pIX Minor structure albumen helps the genome packing greater than 34kb.
Adenovirus by through mediation absorption and mediation endocytosis two kinds not isoacceptor enter host cell. Sick Thereby poison is adsorbed onto cell surface by certain receptors bind of fibrin and the unknown earlier. In the mediation virus The acceptor of swallowing endosome (endosomes) has been accredited as α v integrin (α v integrins). At endocytosis In the process, the acidifying of endosome causes the virus capsid protein conformational change, advances thereby cause virus to break through endosome Enter endochylema, further under the nuclear target signal guidance of coat protein, enter nucleus. The egg of E1 district coding White matter at first raises oneself expression and activates other early stage district's gene expression, and after 8 hours, viral DNA begins Copy, subsequently late protein expression is also carried out the assembling of progeny virion in infected cell. Adenopathy Poison can suppress transcribing and translating of host cell DNA, and promotes that the albumen of self is synthetic. Infected about 30-After 40 hours, the host cell dissolving discharges progeny virion. Clinically, adenovirus generally can be drawn Play the infection of the upper respiratory tract, keratoconjunctivitis sicca, gastroenteritis, pneumonia, bronchitis, hepatitis and cystitis, many Number is slight self-limited disease. Over nearly 20 years, the oral live vaccine of adenovirus is widely used, from making With more than 1,000 ten thousand people's that cross result, evident in efficacy and do not have an obvious toxic and side effect.
2, the characteristics of adenovirus vector
Gene transfer technique and importing approach are the key factors that affects the gene therapy effect. Although reverse transcription Viral vectors is still carrier system most widely used in present transgenosis and the gene therapy, have stablize whole Close the advantages such as expression, but it can only infect the cell of division stage, efficiency of infection is lower, generally can not be used for (in vivo) approach in the body is restricted in the clinical practice unavoidably.
Adenovirus vector is that application and development must be than one of morning aspect gene therapy behind retroviral vector Plant genophore, owing to itself characteristics and carrier system are updated, in recent years in gene therapy Obtain increasingly extensive application. Adenovirus vector at first be applied to the fiber cystis degeneration (Cystic Fibrosis, CF) and the emophysematous gene therapy of alpha-1-antitrypsin deficiency, now be widely used in controlling of other diseases Treat, such as haemophilus A and B, Duehene muscular dystrophy, familial cholesteremia disease, angiopoiesis Postoperative arterial restenosis, Surfactant protein B shortage, erythropoietin(EPO) defective and various tumour, aobvious Show that it has a good application prospect and develops value. Nineteen ninety-five, FDA Food and Drug Administration (FDA) Ratify adenovirus mediated p53 gene therapy and enter clinically, try out the treatment in patients with advanced cancer, receive To significant curative effect, and there is not obvious toxic and side effect.
Adenovirus vector has following characteristics: 1. its host range is wide, and the infection rate height comprises tranquillization Cell and thesocyte; 2. the titre height of virus can reach 109～10
10Plaque forming unit (pfu)/ml, Because its physicochemical property is more stable, can obtain high titre disease by deposition, column chromatography or super centrifugal purification Poisonous carrier; 3. adenovirus does not have after birth, and is insensitive to the deactivation of complement-mediated, more stable in the body; 4. The adenovirus vector of using at present mostly is Ad2 and Ad5, rodent is not had oncogenicity, clinically usually only Cause slight upper respiratory tract reaction, in addition also like this for the immunosupress patient; 5. adenovirus is not usually Be integrated in the host cell gene group, be applicable to that the short-term of gene is expressed, just can not cause the place theoretically The insertion mutation of chief cell, more safer than retrovirus.
3, the construction strategy of adenovirus vector
Present adenovirus carrier derives from adenovirus Ad2 and these two kinds of serotypes of Ad5 mostly.Be applicable to that the adenovirus carrier that gene therapy is used belongs to replication-defective virus.Because E1 encoded protein function is that other all adenoviral gene effective expressions are necessary, therefore modal strategy is exactly with the alternative viral E1 region sequence of foreign DNA, the replication defective adenoviral carrier that produces by the trans E1 of the providing region sequence of packing cell.
Can be used for host's packing cell of the present invention is any host cell that can express adenovirus E 1 district gene, because this cell can provide replication-defective adenoviral to duplicate required E1 district gene expression product.A kind of preferred packing cell commonly used is 293 cells, this cell is transformed through Ad5 adenovirus DNA fragment by HEKC, be loaded with the virogene fragments such as adenovirus E 1 district that are integrated into cellular genome, and continuous expression E1 district albumen, can provide trans compensation for E1 district displaced type adenovirus carrier.
Utilize E3 district disappearance not influence viral infection, cutting the E3 district can increase the exogenous DNA capacity of carrier.Cut E1 and E3 district, foreign gene inserts capacity can reach about 8kb.Clone bigger exogenous dna fragment and then need cut other virus sequence, the function of disappearance is again by complementary cell system or helper virus is trans provides.
The method of recombinant adenovirus of the present invention can produce with several different methods, wherein mainly containing following four kinds of cotransfections, to go into complementary cell system more commonly used by the method that homologous recombination in the body produces recombinant adenovirus, more preferably is to produce with COS/TPC cotransfection homologous recombination method.
(1). extracellular virus dna direct connection method (also claiming the Stow method): this method is to select d1309 adenoviral gene group at the XbaI of 3.7mu (in the E1 district) restriction restriction endonuclease point, cut off and separate the big fragment of 3.7-100mu viral genome (title XbaI fragment), to be loaded with the high order end 0-1.3mu of the adenoviral gene group of foreign DNA then, link on the big fragment of XbaI with ligase enzyme.Connect product and can be used for rotaring redyeing 293 cell with the preparation recombinant adenovirus.
(2). the reorganization of intracellular virus dna homology: very similar with the Stow method, will be loaded with the transfection simultaneously of the big fragment of the adenoviral gene left end 0-1.3/9.8-16mu fragment of foreign DNA and XbaI in 293 cells, recombinating by homology prepares virus vector.
(3). plasmid dna homology reorganization in the cell: this method is utilized the form of plasmid, and terminal linking to each other of the inversion of adenovirus genomic dna formed ring-type, can duplicate in bacterium, thereby simplify the big segmental preparation formality of XbaI.The ring glands viral DNA surpasses the packing limit because bacterial plasmid inserts its total genome length, can not be packaged into virion in cell, and the required fragment of viral DNA homologous recombination only is provided.When making up E1 substituted type adenovirus carrier, at first foreign DNA is inserted in the bacterial plasmid (as p Δ Elsp1A) this plasmid gland-containing virus genome left end sequence (the E1 district that comprises ITR, packaging signal and excalation).Contain the shuttle plasmid of foreign DNA and DNA cotransfection 293 cells of all the other sequences of gland-containing virus genome, produce recombinant adenovirus by intravital homologous recombination.The adenovirus genomic dna that thisly contain all the other sequences, is used for homologous recombination can have different forms, or does not have infectious viral DNA by external through digestion with restriction enzyme, perhaps derives from recombinant plasmid pJM17 or pBHG series plasmid.This method is comparatively commonly used at present, but shortcoming is an also structure ideal not of recombination efficiency.
(4) .COS/TPC cotransfection homologous recombination: each covalent attachment of the two ends of adenovirus DNA has terminal peptide (the Terminal peptide of a 55kDa, TP), being combined with the plaque number that produces behind the adenovirus DNA importing permission cell of terminal peptide is (the Horwitz M.FundamentalVirolgy more than 100 times of naked adenovirus DNA, 2nd edition, edited by Fields B et al.Raven Press, Ltd.NewYork, 1991; 771), show the infection ability important influence of the existence of TP to adenovirus DNA.(Proc.Natl.Acad.Sci.USA.1996 such as Miyake, 93:1320) at first adopt the adenovirus DNA-TP mixture (TPC) that contains TP to carry out homologous recombination, set up the COS/TPC homologous recombination method and prepared recombinant adenovirus, and the recombination efficiency that proves this method can improve nearly a hundred times, and produces parental virus hardly.
(5). homologous recombination in the bacterium: shuttle vectors and adenovirus DNA cotransformation to the bacterium with reorganization ability, as BJ5183, by the homologous recombination in the bacterium, are produced the recombinant dna carrier, the packing by 293 cells then, acquisition recombinant adenovirus.The advantage of this method is to save time, efficiently.
In a preferred embodiment of the invention, use the interior homologous recombination method of bacterium and prepare replication defect type human GM-CSF recombinant adenoviral vector, detailed process is as follows: at first the human GM-CSF bicistronic mRNA is inserted shuttle vectors pShuttle-CMV, place under the control of cytomegalovirus (CMV) promotor, by obtaining the recombinant adenoviral vector of GM-CSF with the homologous recombination of adenovirus DNA carrier pAdEasy-1 in bacterium BJ5183, obtain the replication defective recombinant adenovirus of human GM-CSF at last by the packing of 293 cells, titre reaches 8.67 * 10
10Pfu/ml; And proved prepared human GM-CSF recombinant adenovirus external can effective expression human GM-CSF (100～900ng/10
6Cell/24h).On this basis, with the bi-specific antibody coupling of anti-adenovirus and CD40, the human GM-CSF recombinant adenovirus of preparation CD40 target.
After making human granulocyte macrophage colony stimulating factor recombinant adenovirus of the present invention, just can with the recombinant adenovirus of required purity and optional physiologically acceptable carrier, vehicle or stablizer (Remington ' s Pharmaceutical Sciences, 16 editions, Osol, A., Ed., [1980]), mix with lyophilized form or aqueous solution form, can make the treatment preparation of recombinant adenovirus of the present invention.Acceptable carrier, vehicle or stablizer should be nontoxic under used dosage and concentration for the recipient, and can contain damping fluid such as phosphoric acid salt, Citrate trianion or other organic acid damping fluids; Antioxidant such as vitamins C; Lower molecular weight (less than about 10 residues) polypeptide; Protein such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Amino acid such as glycine, glutamine, l-asparagine, arginine or Methionin; Monose, disaccharides and other carbohydrate such as glucose, seminose or dextrin; Sequestrant such as EDTA; Sugar alcohol such as mannitol or Sorbitol Powder; Salifiable gegenion of shape such as sodium; And/or nonionic surface active agent such as Tween, Pluronics or polyoxyethylene glycol (PEG).As an example, water and physiological saline just can be used as the carrier in the recombinant adenovirus toxin preparation of the present invention.
The replication defect type human GM-CSF recombinant adenovirus of the target of the present invention's preparation has huge using value and advantage, is mainly reflected in following several respects:
(1). Infection in Vitro tumour cell (as the B lymphoma etc.), the tumour knurl seedling of preparation GM-CSF gene transfection carries out the active immunity treatment of tumour, and tumour cell can derive from the fresh knurl body sample or the tumor cell line of tumour patient.
(2). external target sexuality is dyed the antigen presenting cell (as dendritic cell or scavenger cell) that stimulates through tumour antigen, carries out the active immunity treatment of tumour, and wherein dendritic cell can be from peripheral blood of patients monocyte or CD34
+Hematopoietic stem cell expansion.
(3). carrier cells such as Infection in Vitro autologous skin inoblast, by means of carrier cell GM-CSF is imported body, promote hematopoiesis to recover.
(4). inject the enhancing immunity effect with antigen as adjuvant.
(5). directly import in the body by approach in the knurl body or in the body such as infection site direct injection GM-CSF recombinant adenovirus, be used for treatment of diseases such as tumour, infection.
(6) the treatment cost is low, treats than direct injection GM-CSF and hangs down at least 50%, preferably can hang down at least 80%.
(7) frequency injection is few slightly, and only need injection several weeks (as 1-2 week) once usually, thereby reduced the misery of carrying out injection for curing, and easy to use.
(8) long term ground local expression GM-CSF by cells infected and in cell, more approaching with the situation of the GM-CSF of organism merocrine secretion under the physiological condition, thereby can be efficiently and bring into play the effect (especially in the occasion that needs topical application GM-CSF, during) of GM-CSF chronically as oncotherapy.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone of embodiment 1. human GM-CSF cDNA
By the RT-PCR human GM-CSF cDNA that from the activatory human T lymphocyte, increases.The T cellular segregation of at first choosing post (Biotex company) divides from healthy human peripheral blood gets the T cell, external after mitogen ConA (10 μ g/ml, Sigma company) stimulation is cultivated 24 hours, extracts cell total rna with Trizol reagent (Gibco company); With synthetic cDNA first chain of AMV reversed transcriptive enzyme (Promega company), be that template is carried out pcr amplification with it.
Upstream primer and downstream primer sequence that pcr amplification adopts are:
5’cggaattctagaccaccATGTGGCTGCAGAGCCTG?3’(SEQ?ID?NO：4)
5’cgggatccTCACTCCTGGACTGGCTCCC?3’(SEQ?ID?NO：5)
In upstream primer, design EcoRI and XbaI site, designed the BamHI site in the downstream primer.Institute's amplified production comprises whole encoding sequences of human GM-CSF, estimates that size is 456bp.The PCR reaction volume is 50 μ l, and wherein primer concentration is 0.3 μ M, and the amplification parameter is 94 ℃ 1 ', the 58 ℃ capable 2% agarose gel electrophoresis analysis of 1 ', 72 ℃ of 2 ', 20 circulation after product.The PCR product is after EcoRI and BamHI enzyme are cut, directed cloning is to pGEM-3Z carrier (Promega company), connect product transformed into escherichia coli DH5 α (Stratagene company), the recombinant vectors that obtains is designated as pGEM-hGM, with M13-21 and SP6 primer automatically check order (ABI373, PE company).Through sequencing analysis, amplification to human GM-CSF cDNA sequence entirely true, comprise whole encoding sequences (Fig. 1, SEQ ID NO:1 and SEQ ID NO:2).
The clone of embodiment 2.IRES cDNA
With plasmid pIRES-hrGFP (Stratagene company) is that template is carried out pcr amplification.Upstream primer is 5 ' gg GGA TCC cgg GAC GAC TGC ATA GGG TTAC (SEQ ID NO:6), has wherein designed the BamHI site; Downstream primer is 5 ' gGAA TTC CTG CAG CCA TTA TCA TCG TGTTTTTC (SEQ ID NO:7), has wherein designed the EcoRI site; Institute's amplified production estimates that size is 620bp.The PCR reaction volume is 50 μ l, and wherein primer concentration is 0.3 μ M, and the amplification parameter is 94 ℃ 1 ', the 52 ℃ capable 2% agarose gel electrophoresis analysis of 1 ', 72 ℃ of 2 ', 25 circulation after product.The PCR product is after EcoRI and BamHI enzyme are cut, directed cloning is to pBluescript II-KS carrier (Stratagene company), connect product transformed into escherichia coli DH5 α (Stratagene company), the recombinant vectors that obtains is designated as pKS-IRES, with T7 and T3 primer automatically check order (ABI373, PE company).Through sequencing analysis, amplification is to IRES sequence entirely true (corresponding to lowercase part in the middle of Fig. 2) (SEQ ID NO:3).
The structure of 3. pairs of copies of embodiment human GM-CSF fusion gene
At first from the pGEM-hGM carrier, cut out the XbaI/BamHI fragment of GM-CSF, insert pKS-IRES, make GM-CSF cDNA be positioned at the upstream of IRES, obtain recombinant vectors pKS-GM-IRES; From the pGEM-hGM carrier, cut out the EcoRI/SalI fragment of GM-CSF again, insert pKS-GM-IRES, make this GM-CSFcDNA be positioned at the downstream of IRES, obtain the two copy of GM-CSF fusion gene like this, recombinant vectors called after pKS-GM-DC, fusion gene sequence and coded amino acid thereof are seen Fig. 2.
4. pairs of copies of embodiment human GM-CSF construction of recombinant adenovirus containing
The adenovirus shuttle vector pShuttle-CMV that is adopted contains the left and right sides homology arm of Ad5 adenovirus available from Stratagene company, wherein contains a plurality of cloning sites (Fig. 3) for the foreign gene insertion.From the pSK-GM-DC carrier, cut out the NotI/XhoI fragment of two copy GM-CSF fusion genes, be cloned into the NotI/XhoI site of pShuttle-CMV, connect product and transform DH5 α bacterium, obtain recombinant adenovirus shuttle vectors pShuttle-GM-DC.
Recombinant adenovirus shuttle vectors pShuttle-GM-DC is after PacI linearizing and dephosphorylation processing, get pAdEasy-1 carrier (the Stratagene company of 1 μ g and 100ng, Fig. 4) mix, electroporation cotransformation BJ5183 competence bacterium (Stratagene company), homologous recombination (Fig. 5) through the BJ5183 bacterium, obtain the recombinant adenovirus of GM-CSF, positive colony is designated as pAd.GM-DC after the PacI enzyme is cut evaluation.
Positive colony pAd.GM-DC further transforms XL-10 competence bacterium (Stratagene company), carries out a large amount of amplifications of DNA.With high density meat soup TB substratum 500～1000ml positive colony bacterium of increasing in a large number, alkali cracking method extracting cosmid DNA, be dissolved among the 4ml TE, add 44.4g CsCl (Gibco company), 0.4ml ethidium bromide (10mg/ml) mixes, put (the VTi65.1 rotary head of 5ml centrifuge tube (Beckman) gradient centrifugation, 55,000rpm, 20 ℃, 16h), collect the superhelix band,, use the TE dialysed overnight down for 4 ℃ with equal-volume Butanol (Sigma) extracting 4～6 times, or directly add 3 times of volume aseptic deionized waters, 8 times of volume dehydrated alcohols, in 4 ℃ of deposit D NA (3～5 hours), dry back DNA is dissolved in aseptic TE, detects its concentration and purity with spectrophotometer.
The packing of embodiment 5. recombinant adenovirus
Recombinant dna carrier pAd.GM-DC produces human GM-CSF recombinant adenovirus (Fig. 5) through calcium phosphate DNA coprecipitation method rotaring redyeing 293 cell (ATCC company) through born of the same parents' internal packing.Transfection the day before yesterday, treat that 293 cells of transfection are changed fresh culture.In 500 μ l volumes with recombinant adenoviral vector pAd.GM-DC, the CaCl of 5 μ g through the PacI linearization process
2After (final concentration is 0.25M) mixes, add 2 * BBS damping fluid (50mM BES (N, N-bis (2-hydroxyethl)-2-aminoethane-sulfonicacid), 280mM NaCl, the 1.5mM Na of 500 μ l
2HPO
4(pH6.95)), mix rearmounted room temperature effect 10～20 minutes, dropwise add 293 cells, put 37 ℃, 5%CO
2Cultivated 12～24 hours.With 293 cells of cotransfection with mix by different ratioss such as 1: 1,1: 10 and 1: 100 without 293 cells of transfection, be seeded to 96 well culture plates (100 μ l/ hole) respectively, DMEM substratum (50 μ l/ hole) is respectively added in after this 4th day and the 8th day.The cell and the nutrient solution thereof in positive colony hole are collected in after transfection the 12nd ~ 14 day, and the viral supernatant after the ultrasonication is first-generation seed virus (1st seed), in-80 ℃ of preservations.First-generation seed virus is established 2 multiple holes with 293 cells in viral supernatant (10 μ l/ hole) infection 24 well culture plates, each clone; Collected the restriction analysis of the capable cell genomic dna of cell in a multiple hole in 3～4 days, the cell in another multiple hole is collected viral supernatant after ultrasonication, be s-generation seed virus (2nd seed), gives over to amplification and uses.As a result,, in 1: 10 96 well culture plates, collect 95 of 293 cell clones of pathology death, cut through enzyme and identify the recombinant adenovirus clone who obtains two copy GM-CSF at 12～14 days of transfection.
The amplification of embodiment 6. recombinant adenovirus, purifying and titre detect
Enzyme is cut the s-generation seed virus supernatant (2nd seed) of identifying correct recombinant clone, is used to infect 293 cell amplification viruses, after 3～4 days, the ultrasonication cell, centrifugal removal cell debris, viral supernatant is through CsCl gradient centrifugation (25,000rpm, 4 ℃, 2h), spissated viral liquid carry out the CsCl gradient centrifugation second time (35,000rpm, 4 ℃, 3h) purifying, to collect viral liquid at last to containing the PBS damping fluid dialysed overnight of 10% glycerine, packing after the filtration sterilization, quick-frozen are stored in-80 ℃.
Be target cell, detect virus titer with 293 cells with micro-cytopathy political reform.Add 50ml nutrient solution/hole in 96 orifice plates earlier, preceding 8 holes add 25ml through 10
4The viral liquid of dilution, the doubling dilution that begin column is 3 times, totally 11 extent of dilution, each extent of dilution is established 8 multiple holes, every hole adds 50 μ l, 293 cells, and the whole degree of FCS is 5%, establishes the cell contrast simultaneously, after this added the DMEM substratum that 50 μ l contain 10%FCS, result of determination after 12～14 days in the 4th day and the 8th day respectively.One of them positive colony is increased, and the human GM-CSF recombinant adenovirus titre that is increased is 7.67 * 10
10Pfu/ml.Dilute, be distributed into 5 * 10 with water for injection
10Pfu/ml is stored in-80 ℃.
This adenovirus is named as " the reorganization 5 type adenovirus Ad.GM-CSF-DC of two copy expressing human rHuGM-CSFs ", be preserved in Chinese typical culture collection center (China April 11 calendar year 2001, Wuhan), deposit number is CCTCC No.V200103.
The preparation of embodiment 7. anti-adenovirus and people CD40 molecule bi-specific antibody
Mouse anti Ad5 adenoviral fiber protein (fiber) monoclonal antibody 1D6.14 and anti-people CD40 monoclonal antibody G28.5 are respectively from the culture supernatant of its hybridoma cell strain (ATCC company), after the preparation of Protein A (Pharmacia company) affinitive layer purification, with the Fab fragment of papoid (Pierce company) digestion, Protein A (Pharmacia company) affinitive layer purification antibody.The Fab fragment of these two kinds of monoclonal antibodies prepares the bi-specific antibody of anti-Ad5 adenovirus and people CD40 with N-succinimidyl 3-(2-pyridldithio) propionate (SPDP) coupling.Concrete grammar reference literature (Production ofbispecific antibodies.in Current Protocols in Immunology, New York, 1997; P2.13.1).
The preparation of embodiment 8.CD40 targeting gonad virus
Add 10ng/10 in the GM-CSF recombinant adenovirus
6The pfu bi-specific antibody, hatched 1 hour in 4 ℃, bi-specific antibody combines with the GM-CSF recombinant adenovirus by the binding site of its anti-adenovirus, and the binding site of anti-CD40 is wherein still kept in advance, can combine with the people CD40 molecular specificity of surface of cell membrane, so just prepare the human GM-CSF recombinant adenovirus of successful CD40 target, be designated as Ad.GM-CSF/CD40.
The vivoexpression of embodiment 9. human GM-CSF recombinant adenovirus
With recombinant human GM-CSF and IL-4 (Sigma company) cultivator peripheral blood lymphocytes, the preparation dendritic cell.Collect the human dendritic cell of cultivating the 7th day, after abandoning nutrient solution, washing twice, by different superinfection degree (Multiplicity of Infection, MOI) add the recombinant adenovirus of two human GM-CSFs that copy or two copy human GM-CSF recombinant adenovirus of CD40 target, wash after 1 hour and abandon viral liquid, add normal nutrient solution, collect culture supernatant after 48 hours, with ELISA test kit (U.S. R﹠amp; D company) detects GM-CSF content, with ng/10
6Cell/24 hour expression.
Secrete GM-CSF level after the table 1.GM-CSF recombinant adenovirus infected person dendritic cell
Grouping GM-CSF level (ng/10
6Cell/24h)
MOI=5 MOI=20 MOI=100LacZ contrast adenovirus does not detect Ad.GM-DC 49 ± 4.5 163 ± 18 359 ± 13Ad.GM-CSF/CD40 106 ± 11 472 ± 16 889 ± 45
Detected result shows, do not infect do not detect in the culture supernatant of adenovirus dendritic cell GM-CSF (＜5pg/ml), after two copy human GM-CSF recombinant adenovirus (Ad.GM-CSF/CD40) of CD40 target infect dendritic cell, can produce higher levels of human GM-CSF (100-800ng/10 than the recombinant adenovirus (Ad.GM-DC) of the human GM-CSF of two copies
6Cell/24h), and the MOI value of the expression level of GM-CSF when infecting is proportionate, show transgenosis and expression that prepared CD40 target human GM-CSF recombinant adenovirus can more effective Mediated Human dendritic cell, in the immunotherapy of dendritic cell mediation, have using value.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table＜110〉Shanghai Huakang Biological Technology Co. Ltd＜120〉recombinant adenovirus poison and method for making and purposes＜130 of targeting, high expressed human granulocyte macrophage colony stimulus factor〉011975＜160〉7＜170〉PatentIn version 3.0＜210〉1＜211〉456＜212〉DNA＜213〉Homo sapiens＜220〉＜221〉CDS＜222〉(16) .. (450)＜400〉1gaattctaga ccacc atg tgg ctg cag agc ctg ctg ctc ttg ggc act gtg 51
Met?Trp?Leu?Gln?Ser?Leu?Leu?Leu?Leu?Gly?Thr?Val
1 5 10gcc?tgc?agc?atc?tct?gca?ccc?gcc?cgc?tcg?ccc?agc?ccc?agc?acg?cag 99Ala?Cys?Ser?Ile?Ser?Ala?Pro?Ala?Arg?Ser?Pro?Ser?Pro?Ser?Thr?Gln
15 20 25ccc?tgg?gag?cat?gtg?aat?gcc?atc?cag?gag?gcc?cgg?cgt?ctc?ctg?aac 147Pro?Trp?Glu?His?Val?Asn?Ala?Ile?Gln?Glu?Ala?Arg?Arg?Leu?Leu?Asn
30 35 40ctg?agt?aga?gac?act?gct?gct?gag?atg?aat?gaa?aca?gta?gaa?gtc?atc 195Leu?Ser?Arg?Asp?Thr?Ala?Ala?Glu?Met?Asn?Glu?Thr?Val?Glu?Val?Ile45 50 55 60tca?gaa?atg?ttt?gac?ctc?cag?gag?ccg?acc?tgc?cta?cag?acc?cgc?ctg 243Ser?Glu?Met?Phe?Asp?Leu?Gln?Glu?Pro?Thr?Cys?Leu?Gln?Thr?Arg?Leu
65 70 75gag?ctg?tac?aag?cag?ggc?ctg?cgg?ggc?agc?ctc?acc?aag?ctc?aag?ggc 291Glu?Leu?Tyr?Lys?Gln?Gly?Leu?Arg?Gly?Ser?Leu?Thr?Lys?Leu?Lys?Gly
80 85 90ccc?ttg?acc?atg?atg?gcc?agc?cac?tac?aag?cag?cac?tgc?cct?cca?acc 339Pro?Leu?Thr?Met?Met?Ala?Ser?His?Tyr?Lys?Gln?His?Cys?Pro?Pro?Thr
95 100 105ccg?gaa?act?tcc?tgt?gca?acc?cag?act?atc?acc?ttt?gaa?agt?ttc?aaa 387Pro?Glu?Thr?Ser?Cys?Ala?Thr?Gln?Thr?Ile?Thr?Phe?Glu?Ser?Phe?Lys
110 115 120gag?aac?ctg?aag?gac?ttt?ctg?ctt?gtc?atc?ccc?ttt?gac?tgc?tgg?gag 435Glu?Asn?Leu?Lys?Asp?Phe?Leu?Leu?Val?Ile?Pro?Phe?Asp?Cys?Trp?Glu125 130 135 140cca?gtc?cag?gag?tga?ggatcc 456Pro?Val?Gln?Glu<210>2<211>144<212>PRT<213>Homo?sapiens<400>2Met?Trp?Leu?Gln?Ser?Leu?Leu?Leu?Leu?Gly?Thr?Val?Ala?Cys?Ser?Ile1 5 10 15Ser?Ala?Pro?Ala?Arg?Ser?Pro?Ser?Pro?Ser?Thr?Gln?Pro?Trp?Glu?His
20 25 30Val?Asn?Ala?Ile?Gln?Glu?Ala?Arg?Arg?Leu?Leu?Asn?Leu?Ser?Arg?Asp
35 40 45Thr?Ala?Ala?Glu?Met?Asn?Glu?Thr?Val?Glu?Val?Ile?Ser?Glu?Met?Phe
50 55 60Asp?Leu?Gln?Glu?Pro?Thr?Cys?Leu?Gln?Thr?Arg?Leu?Glu?Leu?Tyr?Lys65 70 75 80Gln?Gly?Leu?Arg?Gly?Ser?Leu?Thr?Lys?Leu?Lys?Gly?Pro?Leu?Thr?Met
85 90 95Met?Ala?Ser?His?Tyr?Lys?Gln?His?Cys?Pro?Pro?Thr?Pro?Glu?Thr?Ser
100 105 110Cys?Ala?Thr?Gln?Thr?Ile?Thr?Phe?Glu?Ser?Phe?Lys?Glu?Asn?Leu?Lys
115 120 125Asp?Phe?Leu?Leu?Val?Ile?Pro?Phe?Asp?Cys?Trp?Glu?Pro?Val?Gln?Glu
130 135 140＜210〉3＜211〉1538＜212〉DNA＜213〉GM-CSF＜400〉3gcggccgctc tagaccacca tgtggctgca gagcctgctg ctcttgggca ctgtggcctg 60cagcatctct gcacccgccc gctcgcccag ccccagcacg cagccctggg agcatgtgaa 120tgccatccag gaggcccggc gtctcctgaa cctgagtaga gacactgctg ctgagatgaa 180tgaaacagta gaagtcatct cagaaatgtt tgacctccag gagccgacct gcctacagac 240ccgcctggag ctgtacaagc agggcctgcg gggcagcctc accaagctca agggcccctt 300gaccatgatg gccagccact acaagcagca ctgccctcca accccggaaa cttcctgtgc 360aacccagact atcacctttg aaagtttcaa agagaacctg aaggactttc tgcttgtcat 420cccctttgac tgctgggagc cagtccagga gtgaggatcc cgggacgact gcataggtta 480cccccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 540tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 600cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 660ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 720caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 780ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 840cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 900aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 960tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg 1020gggacgtggt tttcctttga aaaacacgat gataatggct gcaggaattc tagaccacca 1080tgtggctgca gagcctgctg ctcttgggca ctgtggcctg cagcatctct gcacccgccc 1140gctcgcccag ccccagcacg cagccctggg agcatgtgaa tgccatccag gaggcccggc 1200gtctcctgaa cctgagtaga gacactgctg ctgagatgaa tgaaacagta gaagtcatct 1260cagaaatgtt tgacctccag gagccgacct gcctacagac ccgcctggag ctgtacaagc 1320agggcctgcg gggcagcctc accaagctca agggcccctt gaccatgatg gccagccact 1380acaagcagca ctgccctcca accccggaaa cttcctgtgc aacccagact atcacctttg 1440aaagtttcaa agagaacctg aaggactttc tgcttgtcat cccctttgac tgctgggagc 1500cagtccagga gtgaggatcc tctagagtcg acctcgag 1538＜210〉4＜211〉35＜212〉DNA＜213〉＜400〉4cggaattcta gaccaccatg tggctgcaga gcctg 35＜210〉5＜211〉28＜212〉DNA＜213〉＜400〉5cgggatcctc actcctggac tggctccc 28＜210〉6＜211〉30＜212〉DNA＜213〉＜400〉6ggggatcccg ggacgactgc atagggttac 30＜210〉7＜211〉33＜212〉DNA＜213〉＜400〉7ggaattcctg cagccattat catcgtgttt ttc 33
Claims (10)
1. a recombinant adenovirus target, the expressing human rHuGM-CSF, it is characterized in that, the genomic deletion E1 district of this adenovirus and E3 district, and inserted the human granulocyte macrophage colony stimulating factor expression cassette in the E1 zone position, this expression cassette comprises successively: promoter sequence, human granulocyte macrophage colony stimulating factor encoding sequence and polyadenylation signal sequence
And, in the adenovirus surface bonding bi-specific antibody being arranged, this antibody is both specifically at the binding site on adenovirus surface, again specifically at the acceptor molecule of cell surface.
2. recombinant adenovirus according to claim 1 is characterized in that this adenovirus is an Ad5 type adenovirus; And described acceptor molecule is selected from down group: CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule.
3. recombinant adenovirus as claimed in claim 1, it is characterized in that contained promoter sequence comprises the eukaryotic expression promotor that is selected from down group in the human granulocyte macrophage colony stimulating factor expression cassette: cytomegalovirus promoter, bovine papilloma virus promotor.
4. recombinant adenovirus as claimed in claim 1 is characterized in that, the human granulocyte macrophage colony stimulating factor of two copies is arranged in expression cassette.
5. recombinant adenovirus as claimed in claim 4 is characterized in that, this adenovirus is reorganization 5 type adenovirus Ad.GM-CSF-DC, CCTCC No.V200103.
6. a pharmaceutical composition is characterized in that, it comprises the recombinant adenovirus described target of claim 1, the expressing human rHuGM-CSF and the pharmaceutically acceptable carrier or the vehicle of significant quantity.
7. pharmaceutical composition as claimed in claim 6 is characterized in that, this recombinant adenovirus is an Ad5 type adenovirus, and described acceptor molecule is selected from down group: CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule.
8. pharmaceutical composition as claimed in claim 7 is characterized in that, this adenovirus is the reorganization 5 type adenovirus Ad.GM-CSF-DC of expressing human rHuGM-CSF, CCTCC No.V200103.
9. method for preparing recombinant adenovirus target, the expressing human rHuGM-CSF is characterized in that this method comprises:
(a) provide adenovirus DNA-TP mixture,, and be combined with terminal peptide TP at the two ends of adenovirus genomic dna in this adenovirus DNA genomic deletion E1 district and E3 district;
(b) provide gland-containing virus genomic cosmid vector, adenoviral gene group in this cosmid vector has lacked E1 district and E3 district, and inserted the human granulocyte macrophage colony stimulating factor expression cassette in the E1 zone position, wherein this expression cassette comprises successively: promoter sequence, human granulocyte macrophage colony stimulating factor encoding sequence and polyadenylation signal sequence;
(c) the cosmid vector cotransfection in the adenovirus DNA in the step (a)-TP mixture and the step (b) is expressed the host cell of E1 district gene;
(d) screening positive clone;
(e) separation and purification recombinant adenovirus from positive colony.
(f) adenovirus in the step (e) is combined with bi-specific antibody, this antibody is both specifically at the binding site on adenovirus surface, again specifically at the acceptor molecule of cell surface, thereby obtain recombinant adenovirus target, the expressing human rHuGM-CSF.
10. method as claimed in claim 9, it is characterized in that, the cotransfection method is a calcium phosphate DNA co-precipitation infection protocol in step (c), this host cell is 293 cells, and contained promoter sequence is selected from down group in the human granulocyte macrophage colony stimulating factor expression cassette: cytomegalovirus promoter, bovine papilloma virus promotor; Described acceptor molecule is selected from down group: CD1a, CD40, CD44, CD54, CD80, CD83, CD86, MHC molecule; The human granulocyte macrophage colony stimulating factor that two copies are arranged in the expression cassette.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011059729A CN1177053C (en) | 2001-04-13 | 2001-04-13 | Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011059729A CN1177053C (en) | 2001-04-13 | 2001-04-13 | Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1381582A true CN1381582A (en) | 2002-11-27 |
| CN1177053C CN1177053C (en) | 2004-11-24 |
Family
ID=4655032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011059729A Expired - Fee Related CN1177053C (en) | 2001-04-13 | 2001-04-13 | Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1177053C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005071084A1 (en) * | 2003-12-24 | 2005-08-04 | Qijun Qian | Construction of gutted recombinant adenovirus effectively expressing antibodies comprising human constant region and uses thereof |
| WO2005121343A1 (en) | 2004-06-07 | 2005-12-22 | Chengdu Kanghong Biotechnologies Co., Ltd. | Construction of oncolytic adenovirus recombinant specifically expressing immune modulatory factor gm-csf in tumor cells and uses thereof |
| CN101690822B (en) * | 2009-08-13 | 2012-09-05 | 中国科学院苏州纳米技术与纳米仿生研究所 | Medicament carrier for targeted medicament delivery on diseases related to macrophage and preparation method thereof |
| CN106177941A (en) * | 2016-08-09 | 2016-12-07 | 王兴龙 | A kind of adenovirus utilizing expression of GM CSF improves method and the test kit of newcastle inactivated vaccine immune effect |
-
2001
- 2001-04-13 CN CNB011059729A patent/CN1177053C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005071084A1 (en) * | 2003-12-24 | 2005-08-04 | Qijun Qian | Construction of gutted recombinant adenovirus effectively expressing antibodies comprising human constant region and uses thereof |
| WO2005121343A1 (en) | 2004-06-07 | 2005-12-22 | Chengdu Kanghong Biotechnologies Co., Ltd. | Construction of oncolytic adenovirus recombinant specifically expressing immune modulatory factor gm-csf in tumor cells and uses thereof |
| US7951585B2 (en) | 2004-06-07 | 2011-05-31 | Chengdu Kanghong Biotechnologies Co., Ltd. | Construction of oncolytic adenovirus recombinant specifically expressing immune modulatory factor GM-CSF in tumor cells and uses thereof |
| CN101690822B (en) * | 2009-08-13 | 2012-09-05 | 中国科学院苏州纳米技术与纳米仿生研究所 | Medicament carrier for targeted medicament delivery on diseases related to macrophage and preparation method thereof |
| CN106177941A (en) * | 2016-08-09 | 2016-12-07 | 王兴龙 | A kind of adenovirus utilizing expression of GM CSF improves method and the test kit of newcastle inactivated vaccine immune effect |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1177053C (en) | 2004-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1141977C (en) | Improved virus vauines | |
| JPH06508039A (en) | Recombinant defective adenovirus expressing cytokines for antitumor therapy | |
| JP2002539221A (en) | Treatment of cancer with insect cells containing the gene encoded by the recombinant baculovirus | |
| CN1145636A (en) | Vascular endothelial growth factor 2 | |
| JP2011036251A (en) | Virus vector, and use of the same for gene therapy | |
| CN1143386A (en) | Transforming growth factor alpha-H1 | |
| CN101045156B (en) | Special target medicine and its use | |
| CN105899217A (en) | Papaya mosaic virus and virus-like particles in cancer therapy | |
| JPH09510620A (en) | Recombinant adenovirus encoding glial neurotrophic factor (GDNF) | |
| FR2537436A1 (en) | METHOD FOR REGULATING APPETITE AND EFFICIENCY IN THE USE OF FOODS USING INTERFERON | |
| CN1381582A (en) | Targetting high-expression recombinant adenovirus of human granulocyte-macrophage colony stimulating factor and its preparing process and usage | |
| CN1665921A (en) | Adenovirus vectors for immunotherapy | |
| CN1177042C (en) | Recombinant adenovirus of coexpression human P53 gene and human cytokine gene and its preparation method and application | |
| CN1094974C (en) | Recombined adhenovirus expressing human interleukin 12 and its preparation and use | |
| CN1258738A (en) | Recombined adenovirus expressing the stimulating factor of human granulocyte-macrophage colony and its preparation and use | |
| JP4423508B2 (en) | Cancer gene therapy drug | |
| KR20010043764A (en) | Stable gene preparations | |
| CN111909246B (en) | AAV mutants highly efficient in infecting supporting cells | |
| CN1213004A (en) | Recombination leukocyte mesonium-2 adenovirus carrier and producing method | |
| CN1094973C (en) | Recombined adenovirus expressing human interleukin 18 and its preparation and use | |
| CN101033464A (en) | Recombination adenovirus for expressing human particle cytolysin and its preparing method and use | |
| CN1375500A (en) | Thymic peptide fusion protein as one new interferon and its prepn. and use | |
| CN1232536C (en) | Human dry cell factor macrophage clong irritant factor bifunction protein and its preparation | |
| AU717974B2 (en) | Transduced human hematopoietic stem cells | |
| JPH09510358A (en) | Recombinant adenovirus encoding brain-induced neurotrophic factor (BDNF) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: WANG JIANLI Free format text: FORMER OWNER: SHANGHAI HUAKANG BIOLOGICAL TECHNOLOGY CO. LTD. Effective date: 20070511 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20070511 Address after: 200433, room 2, No. 28, Lane 505, Zhongyuan Road, Shanghai, Yangpu District Patentee after: Wang Jianli Address before: 201203 Shanghai Zhangjiang Jing Road No. 351 building 602 room Haitai Patentee before: Shanghai Huakang Biological Technology Co., Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20041124 Termination date: 20160413 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |