WO2008128440A1 - Série de vecteurs recombinés dérivés du virus adéno-associé (raav), leurs procédés de construction et leurs utilisations - Google Patents
Série de vecteurs recombinés dérivés du virus adéno-associé (raav), leurs procédés de construction et leurs utilisations Download PDFInfo
- Publication number
- WO2008128440A1 WO2008128440A1 PCT/CN2008/000835 CN2008000835W WO2008128440A1 WO 2008128440 A1 WO2008128440 A1 WO 2008128440A1 CN 2008000835 W CN2008000835 W CN 2008000835W WO 2008128440 A1 WO2008128440 A1 WO 2008128440A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- raav
- gene
- tumor
- associated virus
- recombinant adeno
- Prior art date
Links
- 238000010276 construction Methods 0.000 title claims abstract description 36
- 239000013603 viral vector Substances 0.000 title claims abstract description 14
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 307
- 241000702421 Dependoparvovirus Species 0.000 claims abstract description 281
- 210000004027 cell Anatomy 0.000 claims abstract description 228
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 226
- 108091007433 antigens Proteins 0.000 claims abstract description 188
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims abstract description 102
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims abstract description 98
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 claims abstract description 91
- 108010072866 Prostate-Specific Antigen Proteins 0.000 claims abstract description 91
- 201000011510 cancer Diseases 0.000 claims abstract description 86
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 claims abstract description 69
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 52
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 52
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 36
- 208000029742 colonic neoplasm Diseases 0.000 claims abstract description 36
- 206010060862 Prostate cancer Diseases 0.000 claims abstract description 32
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims abstract description 32
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims abstract description 30
- 206010061306 Nasopharyngeal cancer Diseases 0.000 claims abstract description 28
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 claims abstract description 27
- 201000011216 nasopharynx carcinoma Diseases 0.000 claims abstract description 27
- 201000007270 liver cancer Diseases 0.000 claims abstract description 20
- 208000014018 liver neoplasm Diseases 0.000 claims abstract description 20
- 208000005718 Stomach Neoplasms Diseases 0.000 claims abstract description 16
- 206010017758 gastric cancer Diseases 0.000 claims abstract description 16
- 201000011549 stomach cancer Diseases 0.000 claims abstract description 16
- 101000998011 Homo sapiens Keratin, type I cytoskeletal 19 Proteins 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 9
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 4
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 4
- 201000005202 lung cancer Diseases 0.000 claims abstract description 4
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 claims description 257
- 239000013598 vector Substances 0.000 claims description 192
- 241000700605 Viruses Species 0.000 claims description 157
- 102000036639 antigens Human genes 0.000 claims description 138
- 239000000427 antigen Substances 0.000 claims description 137
- 238000000034 method Methods 0.000 claims description 100
- 102000007066 Prostate-Specific Antigen Human genes 0.000 claims description 84
- 230000002147 killing effect Effects 0.000 claims description 76
- 102000007469 Actins Human genes 0.000 claims description 56
- 108010085238 Actins Proteins 0.000 claims description 56
- 238000002360 preparation method Methods 0.000 claims description 33
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 29
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 29
- 201000010881 cervical cancer Diseases 0.000 claims description 29
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 29
- 210000001616 monocyte Anatomy 0.000 claims description 23
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 16
- 210000002845 virion Anatomy 0.000 claims description 15
- 206010033128 Ovarian cancer Diseases 0.000 claims description 13
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 13
- 108010066302 Keratin-19 Proteins 0.000 claims description 12
- 201000005243 lung squamous cell carcinoma Diseases 0.000 claims description 12
- 239000002245 particle Substances 0.000 claims description 11
- 101100455063 Caenorhabditis elegans lmp-1 gene Proteins 0.000 claims description 9
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 claims description 9
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims description 9
- 230000003612 virological effect Effects 0.000 claims description 8
- 230000036210 malignancy Effects 0.000 claims description 4
- 210000002540 macrophage Anatomy 0.000 claims description 3
- 102000004641 Fetal Proteins Human genes 0.000 claims description 2
- 108010003471 Fetal Proteins Proteins 0.000 claims description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 210000005075 mammary gland Anatomy 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 abstract description 128
- 101710192602 Latent membrane protein 1 Proteins 0.000 abstract description 69
- 101001034314 Homo sapiens Lactadherin Proteins 0.000 abstract description 65
- 102100039648 Lactadherin Human genes 0.000 abstract description 61
- 230000012010 growth Effects 0.000 abstract description 26
- 238000001727 in vivo Methods 0.000 abstract description 8
- 210000004072 lung Anatomy 0.000 abstract description 8
- 239000012636 effector Substances 0.000 abstract description 2
- 210000000987 immune system Anatomy 0.000 abstract description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 abstract description 2
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 abstract 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 abstract 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 abstract 1
- 101710123134 Ice-binding protein Proteins 0.000 abstract 1
- 101710082837 Ice-structuring protein Proteins 0.000 abstract 1
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 abstract 1
- 102100038358 Prostate-specific antigen Human genes 0.000 abstract 1
- 101710107540 Type-2 ice-structuring protein Proteins 0.000 abstract 1
- 208000019065 cervical carcinoma Diseases 0.000 abstract 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 abstract 1
- 210000000605 viral structure Anatomy 0.000 abstract 1
- 210000004443 dendritic cell Anatomy 0.000 description 228
- 239000013612 plasmid Substances 0.000 description 225
- 238000006243 chemical reaction Methods 0.000 description 186
- 239000002299 complementary DNA Substances 0.000 description 179
- 239000012634 fragment Substances 0.000 description 142
- 238000001514 detection method Methods 0.000 description 133
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 96
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 85
- 239000000047 product Substances 0.000 description 79
- 210000002966 serum Anatomy 0.000 description 77
- 238000012408 PCR amplification Methods 0.000 description 71
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 69
- 101710183399 Keratin, type I cytoskeletal 19 Proteins 0.000 description 66
- 241000701022 Cytomegalovirus Species 0.000 description 63
- 230000029087 digestion Effects 0.000 description 62
- 108091008146 restriction endonucleases Proteins 0.000 description 58
- 239000002609 medium Substances 0.000 description 56
- 239000002773 nucleotide Substances 0.000 description 54
- 125000003729 nucleotide group Chemical group 0.000 description 54
- 108010074328 Interferon-gamma Proteins 0.000 description 50
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 48
- 239000008367 deionised water Substances 0.000 description 48
- 229910021641 deionized water Inorganic materials 0.000 description 48
- 208000015181 infectious disease Diseases 0.000 description 47
- 238000012360 testing method Methods 0.000 description 46
- 230000002829 reductive effect Effects 0.000 description 45
- 102100037850 Interferon gamma Human genes 0.000 description 42
- 239000000872 buffer Substances 0.000 description 42
- 238000000246 agarose gel electrophoresis Methods 0.000 description 41
- 230000034994 death Effects 0.000 description 41
- 231100000517 death Toxicity 0.000 description 41
- 230000003902 lesion Effects 0.000 description 41
- 230000004083 survival effect Effects 0.000 description 41
- 238000009396 hybridization Methods 0.000 description 39
- 239000002246 antineoplastic agent Substances 0.000 description 37
- 229940041181 antineoplastic drug Drugs 0.000 description 37
- 238000003384 imaging method Methods 0.000 description 36
- 238000005516 engineering process Methods 0.000 description 35
- 230000001394 metastastic effect Effects 0.000 description 34
- 206010061289 metastatic neoplasm Diseases 0.000 description 34
- 239000000523 sample Substances 0.000 description 34
- 102100035793 CD83 antigen Human genes 0.000 description 32
- 108010042407 Endonucleases Proteins 0.000 description 32
- 102000004533 Endonucleases Human genes 0.000 description 32
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 32
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 32
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 32
- 230000000694 effects Effects 0.000 description 28
- 210000004698 lymphocyte Anatomy 0.000 description 27
- 230000004544 DNA amplification Effects 0.000 description 26
- 230000001225 therapeutic effect Effects 0.000 description 26
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 24
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 24
- 108010002350 Interleukin-2 Proteins 0.000 description 24
- 102000000588 Interleukin-2 Human genes 0.000 description 24
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 24
- 229910002092 carbon dioxide Inorganic materials 0.000 description 24
- 239000001569 carbon dioxide Substances 0.000 description 24
- 238000004113 cell culture Methods 0.000 description 24
- 239000000463 material Substances 0.000 description 24
- 108020004999 messenger RNA Proteins 0.000 description 24
- 230000036961 partial effect Effects 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- 102000012410 DNA Ligases Human genes 0.000 description 23
- 108010061982 DNA Ligases Proteins 0.000 description 23
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 23
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 22
- 238000001415 gene therapy Methods 0.000 description 20
- 238000002372 labelling Methods 0.000 description 18
- 102000003390 tumor necrosis factor Human genes 0.000 description 18
- 210000001519 tissue Anatomy 0.000 description 17
- 206010067484 Adverse reaction Diseases 0.000 description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 16
- 230000006838 adverse reaction Effects 0.000 description 16
- 238000003149 assay kit Methods 0.000 description 16
- 244000309466 calf Species 0.000 description 16
- 230000022534 cell killing Effects 0.000 description 16
- 230000002596 correlated effect Effects 0.000 description 16
- 230000003247 decreasing effect Effects 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 16
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 15
- 108700019961 Neoplasm Genes Proteins 0.000 description 15
- 102000048850 Neoplasm Genes Human genes 0.000 description 15
- 239000012091 fetal bovine serum Substances 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 15
- 230000006698 induction Effects 0.000 description 15
- 230000008569 process Effects 0.000 description 15
- 238000000926 separation method Methods 0.000 description 15
- 230000003321 amplification Effects 0.000 description 14
- 238000001476 gene delivery Methods 0.000 description 14
- 238000003199 nucleic acid amplification method Methods 0.000 description 14
- 108020004707 nucleic acids Proteins 0.000 description 14
- 102000039446 nucleic acids Human genes 0.000 description 14
- 150000007523 nucleic acids Chemical class 0.000 description 14
- 101000954493 Human papillomavirus type 16 Protein E6 Proteins 0.000 description 12
- 230000005867 T cell response Effects 0.000 description 12
- 239000013608 rAAV vector Substances 0.000 description 12
- 101710181478 Envelope glycoprotein GP350 Proteins 0.000 description 11
- 206010036790 Productive cough Diseases 0.000 description 11
- 210000003802 sputum Anatomy 0.000 description 11
- 208000024794 sputum Diseases 0.000 description 11
- 229930193140 Neomycin Natural products 0.000 description 10
- 239000013599 cloning vector Substances 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 210000005087 mononuclear cell Anatomy 0.000 description 10
- 229960004927 neomycin Drugs 0.000 description 10
- 238000010361 transduction Methods 0.000 description 10
- 230000026683 transduction Effects 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 9
- 239000004677 Nylon Substances 0.000 description 9
- 210000004907 gland Anatomy 0.000 description 9
- 238000001802 infusion Methods 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 229920001778 nylon Polymers 0.000 description 9
- 210000005259 peripheral blood Anatomy 0.000 description 9
- 239000011886 peripheral blood Substances 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 238000007447 staining method Methods 0.000 description 9
- VYZAMTAEIAYCRO-BJUDXGSMSA-N Chromium-51 Chemical compound [51Cr] VYZAMTAEIAYCRO-BJUDXGSMSA-N 0.000 description 8
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 8
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 8
- 108090000695 Cytokines Proteins 0.000 description 8
- 206010019663 Hepatic failure Diseases 0.000 description 8
- 102000008070 Interferon-gamma Human genes 0.000 description 8
- 108090000978 Interleukin-4 Proteins 0.000 description 8
- 108010002586 Interleukin-7 Proteins 0.000 description 8
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 8
- 208000001647 Renal Insufficiency Diseases 0.000 description 8
- 230000001464 adherent effect Effects 0.000 description 8
- 229960000723 ampicillin Drugs 0.000 description 8
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 8
- 230000036528 appetite Effects 0.000 description 8
- 235000019789 appetite Nutrition 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000006143 cell culture medium Substances 0.000 description 8
- 238000002512 chemotherapy Methods 0.000 description 8
- 210000000349 chromosome Anatomy 0.000 description 8
- 238000003776 cleavage reaction Methods 0.000 description 8
- 230000003013 cytotoxicity Effects 0.000 description 8
- 231100000135 cytotoxicity Toxicity 0.000 description 8
- 238000000605 extraction Methods 0.000 description 8
- 239000007850 fluorescent dye Substances 0.000 description 8
- 229940044627 gamma-interferon Drugs 0.000 description 8
- 230000036737 immune function Effects 0.000 description 8
- 238000010212 intracellular staining Methods 0.000 description 8
- 201000006370 kidney failure Diseases 0.000 description 8
- 208000007903 liver failure Diseases 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 210000004976 peripheral blood cell Anatomy 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 238000001959 radiotherapy Methods 0.000 description 8
- 230000007017 scission Effects 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 231100000331 toxic Toxicity 0.000 description 8
- 230000002588 toxic effect Effects 0.000 description 8
- 239000012096 transfection reagent Substances 0.000 description 8
- 241000143437 Aciculosporium take Species 0.000 description 7
- 101150044789 Cap gene Proteins 0.000 description 7
- 238000001976 enzyme digestion Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 229920006284 nylon film Polymers 0.000 description 7
- 101150066583 rep gene Proteins 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 238000004659 sterilization and disinfection Methods 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- 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 6
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 101150055123 afp gene Proteins 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 231100000433 cytotoxic Toxicity 0.000 description 6
- 230000001472 cytotoxic effect Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 238000011895 specific detection Methods 0.000 description 6
- 230000005909 tumor killing Effects 0.000 description 6
- 241000702423 Adeno-associated virus - 2 Species 0.000 description 5
- 108020003215 DNA Probes Proteins 0.000 description 5
- 239000003298 DNA probe Substances 0.000 description 5
- 101150013996 LIP gene Proteins 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 210000000481 breast Anatomy 0.000 description 5
- 230000003211 malignant effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000000717 retained effect Effects 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 3
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 3
- WZFUQSJFWNHZHM-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 WZFUQSJFWNHZHM-UHFFFAOYSA-N 0.000 description 3
- ZRPAUEVGEGEPFQ-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]pyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C=1C=NN(C=1)CC(=O)N1CC2=C(CC1)NN=N2 ZRPAUEVGEGEPFQ-UHFFFAOYSA-N 0.000 description 3
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 3
- 101150045267 CEA gene Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 2
- KZEVSDGEBAJOTK-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[5-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CC=1OC(=NN=1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O KZEVSDGEBAJOTK-UHFFFAOYSA-N 0.000 description 2
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 2
- SXAMGRAIZSSWIH-UHFFFAOYSA-N 2-[3-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,2,4-oxadiazol-5-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NOC(=N1)CC(=O)N1CC2=C(CC1)NN=N2 SXAMGRAIZSSWIH-UHFFFAOYSA-N 0.000 description 2
- IHCCLXNEEPMSIO-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 IHCCLXNEEPMSIO-UHFFFAOYSA-N 0.000 description 2
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 2
- 101150017816 40 gene Proteins 0.000 description 2
- DFGKGUXTPFWHIX-UHFFFAOYSA-N 6-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]acetyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)C1=CC2=C(NC(O2)=O)C=C1 DFGKGUXTPFWHIX-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- -1 CEA Proteins 0.000 description 2
- 206010052358 Colorectal cancer metastatic Diseases 0.000 description 2
- 206010019280 Heart failures Diseases 0.000 description 2
- 208000031220 Hemophilia Diseases 0.000 description 2
- 208000009292 Hemophilia A Diseases 0.000 description 2
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 206010034010 Parkinsonism Diseases 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 210000001787 dendrite Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 201000008752 progressive muscular atrophy Diseases 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 208000002320 spinal muscular atrophy Diseases 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- JQMFQLVAJGZSQS-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JQMFQLVAJGZSQS-UHFFFAOYSA-N 0.000 description 1
- JVKRKMWZYMKVTQ-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]pyrazol-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C=1C=NN(C=1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JVKRKMWZYMKVTQ-UHFFFAOYSA-N 0.000 description 1
- VXZBYIWNGKSFOJ-UHFFFAOYSA-N 2-[4-[5-(2,3-dihydro-1H-inden-2-ylamino)pyrazin-2-yl]pyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC=1N=CC(=NC=1)C=1C=NN(C=1)CC(=O)N1CC2=C(CC1)NN=N2 VXZBYIWNGKSFOJ-UHFFFAOYSA-N 0.000 description 1
- YJLUBHOZZTYQIP-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=N2 YJLUBHOZZTYQIP-UHFFFAOYSA-N 0.000 description 1
- WTFUTSCZYYCBAY-SXBRIOAWSA-N 6-[(E)-C-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-N-hydroxycarbonimidoyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C/C(=N/O)/C1=CC2=C(NC(O2)=O)C=C1 WTFUTSCZYYCBAY-SXBRIOAWSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 102100033668 Cartilage matrix protein Human genes 0.000 description 1
- 206010055114 Colon cancer metastatic Diseases 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000004099 Deoxyribonuclease (Pyrimidine Dimer) Human genes 0.000 description 1
- 101710181046 Endonuclease 3 Proteins 0.000 description 1
- 108010069621 Epstein-Barr virus EBV-associated membrane antigen Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 206010051925 Intestinal adenocarcinoma Diseases 0.000 description 1
- 102000010638 Kinesin Human genes 0.000 description 1
- 108010063296 Kinesin Proteins 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 102100033337 PDZ and LIM domain protein 7 Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 230000010455 autoregulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 101150034144 ci gene Proteins 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000013317 conjugated microporous polymer Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 108700020302 erbB-2 Genes Proteins 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 102000046689 human FOLH1 Human genes 0.000 description 1
- 102000007579 human kallikrein-related peptidase 3 Human genes 0.000 description 1
- 108010071652 human kallikrein-related peptidase 3 Proteins 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000003643 myeloid progenitor cell Anatomy 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 101150012028 tfe gene Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001103—Receptors for growth factors
- A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/00118—Cancer antigens from embryonic or fetal origin
- A61K39/001182—Carcinoembryonic antigen [CEA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
- A61K39/001194—Prostate specific antigen [PSA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
- A61K39/001195—Prostate specific membrane antigen [PSMA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4634—Antigenic peptides; polypeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464403—Receptors for growth factors
- A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/46448—Cancer antigens from embryonic or fetal origin
- A61K39/464481—Alpha-feto protein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/46448—Cancer antigens from embryonic or fetal origin
- A61K39/464482—Carcinoembryonic antigen [CEA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
- A61K39/464494—Prostate specific antigen [PSA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
- A61K39/464495—Prostate specific membrane antigen [PSMA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/464838—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/49—Breast
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/50—Colon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/52—Intestine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/55—Lung
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/58—Prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention relates to vectors and their applications in the field of biology, and in particular to a group of recombinant adeno-associated virus vectors and methods for their construction and their use in the preparation of anti-tumor drugs.
- V adeno-associated virus
- Hermonat et al. described the low-infecting (lip) gene and the cap gene of MV (Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics of adeno- associated virus: isolation and preliminary Characterization of adeno- associated virus type 2 mutants. J Virol. 51: 329-339. Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector : transduction of neomycin resistance into mammalian tissue culture cells Proc. Natl. Acad. Sci. USA 81 : 6466-6470. ). In 1986, Labow et al.
- Non-neoplastic diseases such as Parkinson's syndrome, rheumatoid arthritis, hemophilia, heart failure, progressive muscular atrophy, and Ozheimer's syndrome are mainly used for the treatment of diseases.
- AAV virus used in clinical treatment there are still some problems with the AAV virus used in clinical treatment. For example, the size of the gene for carrying the treatment is significantly limited, the virus itself is unstable, the expression of the therapeutic gene is unstable, and the therapeutic effect is inconsistent.
- the V virus itself is weakly immunogenic, the expressed therapeutic gene is prone to induce an autoimmune response in a patient, and even causes serious side effects.
- AAV is a non-pathogenic defective virus that requires the help of gene products of other viruses (such as adenovirus). Can be assembled into infectious virus particles.
- the AAV genome is about 4700 base pairs (bp) in length, the terminal is a repeating terminal fragment (TR), and the middle is a structural gene of the virus, including the Rep gene associated with viral replication and the viral capsid Cap gene. Due to the instability of the V virus itself and its limited length of carrying exogenous genes (therapeutic genes), it is necessary to carry out recombinant adeno-associated virus (rAAV). A large number of studies have shown that deletion of structural genes in the AAV genome can significantly increase the capacity of exogenous genes.
- rMV adeno-associated virus
- an exogenous gene having therapeutic effects is inserted into rMV, and an infectious rMV virus particle is prepared and injected into a patient to infect an in vivo cell, thereby expressing a therapeutic gene, thereby achieving a therapeutic effect.
- r V is mainly used for the treatment of non-neoplastic diseases such as Parkinson's syndrome, rheumatoid arthritis, hemophilia, heart failure, progressive muscular atrophy, and Ozheimer's syndrome.
- rMV still has some shortcomings, such as unstable virus, low virus titer, and limited capacity to receive therapeutic genes (generally only inserted exogenous gene fragments are about 2000 base pairs (bp), otherwise rMV is stable. Sex will be destroyed). Therefore, it is necessary to design a more rational recombinant adeno-associated virus (rMV) vector to meet the needs of practical applications.
- rMV adeno-associated virus
- the recombinant adeno-associated virus vector provided by the invention comprises the following eight types:
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (V) vector with a prostate-specific antigen PSA (prostate-specific antigen) gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with a prostate specific membrane antigen (PSMA) gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with a chorioembryonic antigen (CEA) gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (V) vector with a keratin-19 (K19) gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with a Her-2/neu antigen gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with an LMP-1 gene of Epstein-Barr virus or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with a breast protein BA46 gene or a mutant gene thereof;
- a recombinant adeno-associated virus vector obtained by replacing an adeno-associated virus structural gene in an adeno-associated virus (AAV) vector with an alpha fetal protein (AFP) gene or a mutant gene thereof.
- AAV adeno-associated virus
- the recombinant adeno-associated virus vector of the present invention is modified on the basis of a known adeno-associated virus vector.
- One one one, - ' is modified on the basis of a known adeno-associated virus vector.
- the gland-associated virus structural gene is a gene, and the tumor specific antigen genes PSA, PSMA, CEA, K19, Her-2/neu, LMP-1, BA46 or AFP are tumor-associated antigen genes, and the mutant gene thereof It is a related tumor-specific antigen gene fragment having the same function.
- the known adeno-associated virus vector has a p5 promoter, and in order to increase the transcription level of the gene of interest, the p5 promoter in the recombinant adeno-associated virus vector can be further replaced with a cytomegalovirus (CMV) promoter, One or several promoters of the beta actin promoter and the SV40 viral promoter.
- CMV cytomegalovirus
- a second object of the present invention is to provide a method for constructing the above recombinant adeno-associated virus vector.
- the construction method provided by the present invention uses a conventional method of genetic recombination, first removing the adeno-associated virus structural gene in the adeno-associated virus vector, and then replacing the knock-out gene with the specific antigen gene or its mutant gene to obtain a recombination.
- Adeno-associated virus vector Adeno-associated virus vector.
- the p5 promoter in the recombinant adeno-associated virus vector may be further replaced with a macrophage virus promoter and beta actin. Promoter and one or several promoters of the SV40 viral promoter.
- Products related to the PSA recombinant adeno-associated virus vector of the present invention including recombinant adeno-associated virus plasmids, recombinant adeno-associated virus particles, and cell lines infected or transfected with the recombinant adeno-associated virus vector of the present invention, such as monocyte-macrophages - a dendritic cell line, a T lymphocyte cell line, etc.
- the related gene in the recombinant adeno-associated virus vector - a prostate specific antigen gene or a mutant gene thereof may be in a monocyte-macrophage-dendritic cell line or It is within the scope of the present invention to obtain expression in a cell line such as a T lymphocyte cell under the action of a transcriptional promoter.
- Another object of the present invention is to provide an antitumor drug.
- the active ingredient of the antitumor drug provided by the present invention is the above recombinant adeno-associated virus vector or a product related to the recombinant adeno-associated virus vector of the present invention.
- a tumor antigen-wild-type and/or mutant tumor-specific antigen gene is introduced into a monocyte-macrophage-dendritic cell line, and dendritic cells are induced,
- a tumor antigen-wild-type and/or mutant tumor-specific antigen gene is introduced into a monocyte-macrophage-dendritic cell line, and dendritic cells are induced.
- cytotoxic T lymphocytes stimulated by the dendritic cells such as but not limited to T lymphocytes and B lymphocytes
- the related malignant tumors are prostate cancer, epithelial cell malignant tumor, breast cancer, colon cancer, gastric cancer, lung adenocarcinoma, lung cancer, ovarian cancer, nasopharyngeal cancer, cervical cancer, lung squamous cell carcinoma, liver cancer and the like.
- the medicament provided by the present invention may be in the form of a solvent or a powder.
- the solvent can be selected in a variety of ways, such as cell culture fluid (basic), physiological saline or phosphate buffer.
- One or more pharmaceutically acceptable carriers may also be added to the above drugs as needed.
- the carrier includes conventional diluents, absorption enhancers, surfactants and the like in the pharmaceutical field.
- the method of administration may be to first isolate the monocytes in the tumor patient, and then infect or transfect the mononuclear cells of the patient. Or mature dendritic cells transformed with wild-type and/or mutant prostate-specific antigen PSA Stimulation of cytotoxic tau lymphocytes is returned to tumor patients.
- the above-mentioned drugs are generally used in an amount of 100 ⁇ 1/5 X 10 6 / each time, twice a month, and the course of treatment is usually 6 months. Dosage and treatment can be adjusted according to the actual situation.
- the medicament of the present invention can also be combined with antibiotics, immunostimulants and tumor-targeted drugs.
- the invention also provides a method of killing a tumor.
- the method for killing tumor provided by the present invention may include the following steps:
- Mononuclear-macrophage-dendritic cells or sputum lymphocytes naturally produced in a system in which a tumor is present are carried by the present invention to carry a wild-type tumor-specific antigen a recombinant adeno-associated virus vector of the gene and/or a recombinant adeno-associated virus vector carrying the mutant specific antigen gene is infected or transfected, or treated with a product associated with the recombinant adeno-associated virus vector of the present invention, each of which obtains the treated cell;
- step 2) adding the monocyte-macrophage-dendritic cells treated in step 1) to the tumor system to kill the tumor; or treating the untreated sputum lymphocytes with the treated monocytes - Macrophage-dendritic cells are mixed and cultured to form antigen-specific cytotoxic sputum lymphocytes, and the antigen-specific cytotoxic sputum lymphocytes are added to the tumor system to kill the tumor; or the treated sputum lymphocytes and The treated monocyte-macrophage-dendritic cells are added to the tumor system to kill the tumor.
- the method for killing tumor of the present invention can be specifically applied to tumor treatment, comprising administering a tumor patient to return an antigen-specific cytotoxic sputum lymphocyte, which is derived from a naturally occurring sputum lymphocyte derived from a patient.
- the patient's monocyte-macrophage-dendritic cells are produced by mixed culture. Prior to mixed culture, these monocyte-macrophage-dendritic cells have been infected with a recombinant adeno-associated virus carrying a wild-type specific antigen gene and/or a recombinant adeno-associated virus vector carrying a mutant specific antigen gene of the present invention. Or transfection, or treatment with a product associated with a recombinant adeno-associated viral vector of the invention;
- a tumor patient is administered a monocyte-macrophage-dendritic cell derived from the patient.
- these monocyte-macrophage-dendritic cells Prior to reinfusion, these monocyte-macrophage-dendritic cells have been infected with a recombinant adeno-associated virus carrying a wild-type specific antigen gene and/or a recombinant adeno-associated virus vector carrying a mutant specific antigen gene of the present invention.
- transfection, or treatment with a product associated with a recombinant adeno-associated viral vector of the invention
- a tumor patient is administered a reinfusion of the above-described patient-derived lymphocytes and naturally occurring monocyte-macrophage-dendritic cells derived from the patient.
- these sputum lymphocytes Prior to reinfusion, these sputum lymphocytes have been infected or transfected with a recombinant adeno-associated virus carrying a wild-type specific antigen gene and/or a recombinant adeno-associated virus vector carrying a mutant specific antigen gene, or recombined with the present invention.
- Adeno-associated virus vector related product processing is provided to produce a recombinant adeno-associated virus carrying a wild-type specific antigen gene and/or a recombinant adeno-associated virus vector carrying a mutant specific antigen gene, or recombined with the present invention.
- the present invention provides a recombinant adeno-associated virus (rAAV) vector having high stability and a large capacity carrying a foreign gene (PSA).
- the structural gene of V is ⁇ 3 ⁇ 4 and Zi/(3 ⁇ 4p gene is wild-type or mutant tumor-specific antigen genes PSA, PSMA, CEA, K19, Her-2/neu, LMP-1, BA46 or AFP replaced.
- the recombinant adeno-associated virus vector of the present invention can transport the specific antigenic antigen of the wild-type or mutant type carried by it into the monocyte-macrophage-dendritic cell line, carrying the specific antigen gene
- the cells are used to stimulate effector cells of the immune system (not limited to T lymphocytes and B lymphocytes).
- the dendritic cells infected with the rMV of the present invention and the induced cytotoxic T lymphocytes can effectively inhibit the growth of the relevant malignant tumor cells or kill the tumor cells in the patient, and thus, the recombinant gland correlation of the present invention
- Viral vectors or products associated with the recombinant adeno-associated viral vectors of the invention can be used to prepare anti-tumor drugs.
- the invention has important theoretical and practical significance in the clinical treatment and application of malignant tumors, and has broad application prospects.
- Figure 1 is a schematic diagram showing the structure of a recombinant adeno-associated virus vector.
- Figure 2A to Figure 2H show the results of restriction enzyme digestion and PCR detection of eight recombinant adeno-associated virus vectors rAAV.
- Figure 3 is a flow chart showing the preparation of recombinant adeno-associated virus rAAV.
- Figure 4A shows the results of virus titer detection of recombinant adeno-associated virus rAAv/PSA
- Figure 4B shows the results of virus titer detection of recombinant adeno-associated virus Raav/PSMA
- Figure 4C shows the viruses of recombinant adeno-associated virus rAAV/CEA and rAAV/K19
- Figure 4E shows the results of virus titer detection of recombinant adeno-associated virus rAAV/AmHer-2/neu
- Figure 4F shows the results of virus titer detection of recombinant adeno-associated virus rMV/LMP-1
- Figure 4G shows recombinant gland The virus titer detection result of the related virus rAAV/BA46
- Fig. 4H shows the virus titer detection result of the recombinant adeno-associated virus rMV/AFP.
- Figure 5 shows the experimental procedure for killing tumors based on mononuclear cells of one or more rAAV virus-infected tumor patients carrying tumor antigen genes.
- Figure 6A shows the results of the detection of the efficiency of recombinant adeno-associated virus rMV/PSA infection of peripheral blood mononuclear cells
- Figure 6B shows the results of the detection of the efficiency of recombinant adeno-associated virus rMV/PSMA infection of peripheral blood mononuclear cells
- Figure 6C shows the recombinant adeno-associated virus rMV /CEA infection test results of peripheral blood mononuclear cells
- Figure 6D shows the efficiency of detection of recombinant adeno-associated virus r V/K19 infection of peripheral blood mononuclear cells
- Figure 6E shows recombinant adeno-associated virus rAAV/AmHer-2/neu infection
- Figure 6G shows the infection of peripheral blood mononuclear cells
- Figure 7A shows the results of detection of CD80, CD83 and CD86 levels in DCs infected with rAAV/PSA recombinant adeno-associated virus, respectively;
- Figure 7B shows the results of detection of CD80, CD83 and CD86 levels in DCs infected with recombinant adeno-associated virus rMV/PSMA
- Figure 7C shows the results of detection of CD80, CD83 and CD86 levels in DCs of recombinant adeno-associated virus r V/CEA infection;
- Figure 7D shows the results of CD80, CD83 and CD86 levels in DCs infected with recombinant adeno-associated virus rAAV/K19;
- Figure 7E shows the results of detection of CD80, CD83 and CD86 levels in DCs infected with recombinant adeno-associated virus rMV/BmHer-2/neu;
- Figure 7F shows DC expression of CD80, CD83 and CD86 in recombinant
- Figure 8A shows the results of detection of IFN- ⁇ expression levels of CTLs induced by DCs infected with r V/PSA recombinant adeno-associated virus;
- Figure 8 shows IFN-induced CTLs induced by DCs infected with r V/PSMA recombinant adeno-associated virus.
- Figure 8C shows the results of IFN- ⁇ expression levels of CTLs induced by rAAV/CEA-infected DCs
- Figure 8D shows the results of IFN- y expression levels of CTLs induced by r V/K19-infected DCs
- Figure 8E shows the results of IFN- y expression levels of CTLs induced by rMV/CmHer-2/neu-infected DCs
- Figure 8F shows the results of IFN- y expression levels of CTLs induced by rAAV/LMP-1 infected DCs
- Figure 8G shows the results of IFN- ⁇ expression levels of CTLs induced by rAAV/BA46-infected DCs
- Figure 8H shows the results of IFN- ⁇ expression levels of CTLs induced by rAAV/AFP-infected DCs.
- Figure 9A shows the results of detection of IFN- ⁇ expression levels of CTLs induced by DCs infected with rMV/PSA recombinant adeno-associated virus;
- Figure 9 shows CTL killing tumor cells induced by rMV/PSMA-infected DCs and killing specificity test results
- Figure 9C shows the results of CTL killing tumor cells and killing specificity induced by r V/CEA-infected DCs;
- Figure 9D shows CTL killing tumor cell assays and killing specific detection results induced by r V/K19-infected DCs;
- Figure 9E shows CTL killing tumor cell assay and killing specificity test results induced by rAAV/AmHer-2/neu-infected DC;
- Figure 9F shows CTL killing tumor cell assay and killing specificity induced by rMV/LMP-1 infected DC;
- Figure 9G shows the CTL killing tumor cell test and killing specific test results induced by rAAV/BA46-infected DC;
- FIG. 10A is an imaging observation result of a change of metastatic lesions before and after CTL treatment induced by rAAV/PSA-infected DC
- FIG. 10B is an image of a change of metastatic lesions before and after CTL treatment induced by rMV/PSMA-infected DCs
- Figure 10C is an imaging observation of a change in metastatic lesions before and after CTL treatment induced by rMV/CEA-infected DCs in a colon cancer patient
- Figure 10D is a case of cervical cancer patients induced by rMV/K19-infected DCs.
- Figure 10E shows changes in metastatic lesions before and after CTL treatment in a breast cancer patient infected with rAAV/AmHer-2/neu and r V/CmHer-2/neu Imaging findings of the situation;
- Figure 10F is an imaging observation of a change in metastatic lesions before and after CTL treatment induced by rAAV/LMP-1 infected DCs in patients with nasopharyngeal carcinoma;
- Figure 10G is a case of breast cancer patients undergoing r V Imaging findings of changes in metastatic lesions before and after CTL treatment induced by /BA46-infected DCs.
- Figure 11A shows the changes of serum PSA tumor antigen levels in four patients before and after CTL treatment induced by rAAV/PSA-infected DCs.
- Figure 11B shows the serum levels of four patients before and after CTL treatment induced by rAAV/PSMA-infected DCs. Changes in PSMA tumor antigen levels;
- Figure 11C shows changes in serum CEA tumor antigen levels in patients with metastatic colorectal cancer who were induced by rAAV/CEA-infected DCs before and after CTL treatment;
- Figure 11D shows four cases of K19-positive lungs. Patients with squamous cell carcinoma before and after CTL treatment induced by rAAV/K19-infected DC Changes in serum CK19 tumor-associated antigen levels;
- Figure 11E shows four breast cancer patients
- the percentage concentrations are mass/volume (w/v) percent concentration or volume/volume (v/v) percent concentration unless otherwise stated.
- Example 1-1 Construction and detection of recombinant adeno-associated virus vectors rAAV/PSA and rAAV/mPSA (sudden-variant)
- A. pBR322 plasmid carrying V 2 type whole genomic DNA (designated pBR-V2 ): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Bower Gene International Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector : transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81 : 6466-6470.
- Human primary prostate cancer cells Isolated from cancer tissues of prostate cancer patients or obtained from commercial sources, immunohistochemistry confirmed prostate-specific antigen positive.
- the pCI-ne plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid carrying the SV40 early promoter, PSG424, was purchased from Clonitic, USA.
- the recombinant adeno-associated virus vector carrying the prostate-specific antigen (PSA) gene or its mutant gene of the present invention is constructed by the following method (as shown in Fig. 1), and the specific process comprises the following steps: 1. Recombinant gland-related Construction of viral vectors
- the nucleotide sequence containing the restriction enzymes EcoR I and EcoR V cleavage site was inserted into the plasmid.
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and EcoR N, 10 IU.
- T 4 DNA ligase purchased from Promega, USA
- 1.5 ⁇ l 10 XT 4 DNA ligation buffer and 11.5 ⁇ l of deionized water reaction conditions were: water bath at 4 ° C for 8 hours.
- the complete TR sequence at both ends is retained or the 75th nucleotide sequence of the TR at both ends of the V genome is inserted into a fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability and increase of the rAAV virus.
- the replication efficiency of the virus was as follows: First, the restriction endonuclease I (purchased from Promega, USA) was used to cleave the TR at both ends, and the reaction system was: lwg the plasmid prepared above, 10 U Ban I, 1.5 ⁇ l 10X buffer G and 11.5 ⁇ 1 deionized water; reaction conditions: water bath at 37 ° C for 4 hours, then insert 9 nucleotide fragments into the plasmid, the reaction system is: 500ng plasmid, 300ng 9 nucleotide sequence, 10IU T 4 DNA ligase (purchased from Promega, USA), 1.5 ⁇ l 10XT 4 DNA ligation buffer and 11.5 ⁇ l of deionized water; reaction conditions were: water bath at 4 ° C for 8 hours.
- Amplification of the CMV promoter, SV40 early promoter by gene amplification technology (polymerase chain reaction, PCR).
- the specific method is as follows: Firstly, the CMV promoter was PCR-amplified under the guidance of primer 1: AGATCTTCAATATTGGCCAT and primer 2: TGTCAGAAGCACTGACTGC with pCI-neo plasmid (purchased from Promega, USA).
- the PCR amplification conditions were as follows: 94 ° C 4 minutes; further 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute, a total of 30 cycles; the last 72 ° C for 8 minutes, after the reaction, the PCR product was tested by 1.2% agarose gel electrophoresis An expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; 'C 30 sec, 60 ⁇ 35 sec, 72 ° C 40, sec, 30 cycles in total; last 72 ⁇ 8 min, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, and an expected 359 bp appeared. A specific band, which was recovered and purified to obtain an SV40 early promoter.
- PCR amplification conditions are: first 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 58 ° C for 35 seconds, 72 ° C for 1 minute and 20 seconds, a total of 30 cycles; finally 72 ° C for 8 minutes, After the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis, and a desired specific band appeared at 1176 bp, and the target band was recovered and purified to obtain a beta actin promoter.
- the rRNA was reverse transcribed into its cDNA and used as a template.
- the full-length PSA cDNA was amplified by PCR under the guidance of primer 7: ATTCCGCCGGAGAGCTGTG and primer 8: CCCAGGACACAGAGAGAGGAC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; 94 ° C 30 seconds, 59 ⁇ 35 seconds, 72. C 1 minute 15 seconds, a total of 30 cycles; the last 72 ⁇ 8 minutes, after the end of the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, and an expected specific band appeared at 1006 bp.
- PSA cDNA fragment A (primer 9: ATGTGGGTCCCGGTTGTCTTC and primer 10: TGTGGCCGACCCAGCAAG), PSA cDNA fragment B (primer 11: GCCTGTTTCATCCTGAAG and primer 12: GAGCTCGGCAGGCTCTGAC), C were obtained by the same method as above.
- primer 13 ACGGATGCTGTGAAGGTC and primer 14: AGCACCTGCTCGGGTGATTC)
- D primer 15: GGTGCTCAGGGGTTGGCCAC).
- step D Insert the above amplified CMV promoter, SV40 early promoter, beta actin promoter, full-length PSA cDNA or partial PSA cDNA fragment into the P BR-MV2 vector modified in step A by DNA ligation technology.
- the first digestion reaction is carried out, and then the ligation reaction is carried out, wherein the enzyme digestion reaction system is: lg plasmid; 10 U restriction endonucleases rf l and 53 ⁇ 47 ⁇ (purchased from Promega, USA), 2. 5 ⁇ 1 10 X buffer C and 19.
- the ligation reaction system is: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions: water bath at 4 ° C for 8 hours. Then, the plasmid carrying the promoter and the full-length PSA cDNA were digested with restriction enzymes EcoR I and EcoR V, respectively. The system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector (designated rAAV/PSA) carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length PSA cDNA, and the CMV promoter carrying the SV40 early promoter were obtained.
- a beta actin promoter and a recombinant adeno-associated viral vector of a different PSA cDNA fragment (mutant) of A or B or C or D designated rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and r V/DmPSA, respectively
- rAAV/mPSA Uniformly named rAAV/mPSA
- the ligated DNA-r V/PSA and rAAV/mPSA were separately introduced into genetically engineered E. coli) DH5 competent cells (Ir itrogen, USA), and resistant to LB plates containing 10 ( ⁇ g/mL ampicillin). Sex screening, single colonies were picked, plasmids were extracted and purified to obtain rAAV/PSA plasmid and rAAV/mPSA plasmid.
- the purified r V/PSA plasmid and the rAAV/mPSA plasmid obtained in the first step were digested with restriction enzymes (Nsi I, EcoR V and EcoR I, Not I and Bam W, Pst I in sequence).
- the restriction enzymes used were purchased from Promega, USA.
- the r V vector without PSA gene was used as a negative control (CMV)
- CMV negative control
- the promoter, the SV40 early promoter, and the beta actin promoter are sequentially inserted into the modified pBR-V2 vector of step A, and the vector is digested with restriction endonuclease Hind YLI. After the reaction, the product is digested.
- the assay was performed by 1.2% agarose gel electrophoresis, and the detection result of the r V/PSA plasmid is shown in Fig. 2A (1. DNA molecular weight standard. 2. rAAV vector without PSA gene (#i/ ⁇ III endonuclease) 4. r AAV/PSA (vfe I endonuclease) 5. rk /?Sk (EcoR V and EcoR I endonuclease) 6. rAAV/PSA (A3 ⁇ 4i I and BanR I endonuclease). . rMV/PSA I endonuclease). The specific band of 6. 2 kb was digested by I, and the specific bands of 1.
- the PCR results of the rMV/mPSA plasmid were also consistent with the expected results (the size of the amplified product was 240 bp, 171 bp, 228 bp, 147 bp in sequence).
- the above results indicated that the recombinant adeno-associated virus vector rAAV/PSA carrying the prostate specific antigen (PSA) gene and the recombinant adeno-associated virus vector rAAV carrying the prostate specific antigen (PSA) mutant gene were obtained with the correct insertion position and sequence.
- PSA prostate specific antigen
- PSA prostate specific antigen
- Example 1-2 Preparation of recombinant adeno-associated virus (rAAV) and determination of virus titer
- Example 1-1 Recombinant adeno-associated virus vector rAAV/PSA carrying prostate specific antigen (PSA) gene and recombinant adeno-associated virus vector carrying a prostate specific antigen (PSA) mutant gene rAAV/mPSA (rAAV/ AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA).
- PSA prostate specific antigen
- AAV-HEK293 cells containing adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the cell chromosome: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva) Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Inter Ivery into human dendrit ic cells by an adeno-associated virus vector. Cancer Gene Therapy 8 : 948-957. ).
- Liposomal transfection reagent Lipofecti was purchased from Invotrogen, USA.
- F. PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 10.1 C m cell culture dish, for example, when V-HEK293 cells were grown in a carbon dioxide cell incubator to about culture.
- the dish area is 70%, proceed as follows:
- Lipofectin 1.0 ⁇ g AAV/PSA or rMV/mPSA, 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50. 0 ⁇ 1 5% fetal bovine serum (or calf serum) DMEM medium was mixed and allowed to stand at room temperature for 20 minutes.
- rAAV/PSA rAAV/AmPSA
- rAAV/BmPSA partial PSA cDNA fragments
- the virus titer of various rAAV viruses (rAAV/PSA, rAAV/AraPSA.rAAV/BmPSA, rAAV/CmPSA, rAAV/DmPSA) obtained in the first step is determined by conventional dot blot hybridization, and the specific method includes the following steps:
- the DNA probe used is a specific probe for the PSA gene.
- the nylon membrane was placed in a dot blotter, and the alkali-denatured rAAV virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- D Prepare a DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is "PSA cDNA obtained in Step 1-1 of Example 1-1. After PCR amplification, PCR amplification The product was subjected to 1.2% agarose gel electrophoresis, and the PCR amplification product was detected under ultraviolet light, and a positive band appeared, indicating that the probe was successfully labeled.
- the detection results of rAAV/PSA are shown in Fig. 4A, and the virus titer of rAAV/PSA is 10 12 - 10 11 copies/Ml o.
- the virus titer of four rAAV/mPSA can also reach 10 12 - 10 11 copies / mL.
- Examples 1-3 tumor antigen-inducing monocyte-macrophage-dendritic cell line killing tumor experiment materials and sources:
- rAAV virus rAAV/PSA and rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA) 0
- AIM-V cell culture medium purchased from H Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the entire process of infecting a monocyte-based tumor killing experiment of a tumor patient with a rAAV virus carrying a prostate specific antigen gene and a mutant gene thereof comprises the following steps:
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- Example 1-2 of the present invention Add one (or more, better effect)
- the rAAV virus obtained in Example 1-2 of the present invention is added in an amount of about 100-1000 M0I, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- the old medium was removed, and the AIM-V medium containing GM-CSF, IL-4 (800 IU/mL) and TNF-a (20 IU/mL) was added, and the culture was continued.
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- a specific fluorescent antibody against a tumor-associated antigen-prostate-specific antigen (purchased from BD, USA) is used to label the mononuclear cells infected with the r V of the present invention or Immature DCs were then flow cytometry to detect the number of positive cells.
- PSA tumor-associated antigen-prostate-specific antigen
- Tumor antigens of rAAV are approximately 90% efficient in infecting peripheral blood mononuclear cells, ie approximately 90% of peripheral blood mononuclear cells can be
- the rAAV virus infection demonstrated that the rAAV of the present invention has a high infection efficiency.
- DC dendritic cells
- the levels of DC expression CD80, CD83 and CD86 were positively correlated with the function of DC.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- Stimulated DCs and non-stimulated DCs were controls.
- the results of detection of CD80, CD83 and CD86 levels of DCs of recombinant adeno-associated virus rAAV/PSA infection are shown in Fig. 7A, and the levels of CD molecules expressed by DCs infected with rAAV/PSA were significantly higher than those of the control.
- rAAV/mPSA (rAAV/AmPSA, rMV/BmPSA, rAAV/CmPSA and rAAV/DmPSA)
- the infected DC was also significantly higher than the control. It was demonstrated that the DCs induced by the tumor-associated antigen-prostate-specific antigen and its mutant r V infected peripheral blood mononuclear cells were strongly functional.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN-y induced by DCs infected with the rMV of the present invention was detected by a method similar to that of Step A (DCs induced by PSA protein-stimulated DCs and non-stimulated DCs as controls), DC and peripheral blood. After the mixed culture of lymphocytes, the cells were harvested and labeled with fluorescent staining by conventional intracellular staining. The antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry. .
- the expression level of IFN- ⁇ of CTL induced by rAAV/PSA-infected DC is shown in Fig. 8A, and the level of IFN- ⁇ expressed by CTL induced by rAAV/PSA-infected DC is significantly higher than that of the control.
- r V/mPSA rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA
- CTL Cytotoxicity T lymphocyte
- the cytotoxic T lymphocytes induced by the rAAV/PSA-infected DC in step 1 were mixed with prostate cancer cells by 20:1 (lymphocytes: tumor cells), and the conventional MTT method and 51 Cr were used. (Chromium-51) killing test, detecting the activity of CTL killing tumor cells.
- the statistical results of the tumor cell killing rate of the CTL induced by the DCA/PSA-infected DC are shown in Fig. 9A, and are induced by the DCA-infected DC carrying the tumor antigen-prostate specific antigen and its mutant form constructed and prepared by the present invention.
- CTL can be more effectively lysed (killed)
- Prostate cancer cells are isolated from four prostate cancer patients (A, B, C, D) with a kill rate of 43-52%. Similar killing rates were obtained for CTLs induced by DCs infected with rAAV/mPSA (rAAV/AmPSA, rAAV/BmPSA, rAAV/CmPSA and rAAV/DmPSA).
- rAAV cytotoxic T lymphocytes induced by infected DCs to kill tumor cells.
- BmPSA, rAAV/CmPSA and rAAV/DmPSA The specificity of cytotoxic T lymphocytes induced by infected DCs to kill tumor cells.
- the tumor cell killing specificity test result of the CTL induced by the DCA/PSA-infected DC is as shown in FIG. 9A, and the tumor-associated antigen-prostate-specific antigen and its mutant rAAV infected by the present invention are prepared and prepared.
- the CTL induced by DC has no killing effect on lung, breast, liver and kidney cells, and proves that the tumor-associated antigen-prostate specific antigen constructed and prepared by the present invention CTLs induced by their mutant rAAV-infected DCs are antigen-specific, ie, have no killing effect on antigen-negative cells.
- rMV-DC tumor-bearing antigen-prostate specific antigen and its mutant rAAV-infected DC
- Example 1-4 clinical trial of tumor treatment First, the efficacy and survival time detection
- Recombinant adeno-associated virus-dendritic cell technology which is the DC (rMV) infected with rAAV (r AAV/PSA, rAAV/AmPSA rMV/BmPSA, rAAV/CmPSA and rMV/DmPSA) of the present invention, respectively, in Examples 1-3 - DC) induced CTL reinfusion in 20 patients with prostate cancer, the infusion volume was 1 X 10 9 - 5 X 10 9 .
- Treatment course usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to every 1 - 3 months of treatment.
- Therapeutic effect (reaction after rAAV-DC treatment) The statistical results are shown in Table 1-1 (B: serum tumor markers decreased or disappeared. Q: The patient's quality of life improved. If the pain is reduced or disappeared, the appetite is increased, etc. C : CT or PET-CT shows that the cancer lesions or metastatic lesions are significantly reduced or disappeared.), Adverse reactions: In most cases, mild flu-like reactions occur within a short period of time after treatment, but the patients can withstand, and the symptoms disappear in a short period of time. No serious adverse reactions and toxic reactions were observed.
- PSA mutant PSA cDNA fragment D 18 26 B, Q, C
- PSA mutant PSA cDNA fragment D 12 20 Q, C
- Tumor type Number of cases Total Average course Total mean Time to live Number of cases of prostate cancer 20 219 10. 95 368 18. 4 4
- the imaging findings of 20 cases of prostate cancer patients before and after treatment were observed.
- the imaging findings of a case of metastatic lesions before and after CTL treatment induced by DCs infected with rAAV/PSA in stage IV are shown in Fig. 10A.
- rAAV-DC rAAV-infected DC
- the CTL induced by DC can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients, and can be used for preparing antitumor drugs.
- Fig. 11A The change of the data (the data from the test results of the experimental hospital) is shown in Fig. 11A.
- the serum antigen PSA level in the serum was significantly decreased after the CTL treatment induced by the rAAV-infected DC (rAAV-DC) of the present invention. It indicates that the tumor burden in the patient is significantly reduced (the tumor cells are significantly reduced), further demonstrating that the CTL induced by the DC infected with the rMV of the present invention can effectively inhibit the growth of malignant cells or kill tumor cells, and can be used for Preparation of anti-tumor drugs.
- Part II Recombinant adeno-associated virus vector rAAV/PSMA
- Example 2-1 Recombinant Adeno-associated Virus Vector Construction and Detection of rMV/PSMA and rMV/mPSMA Materials and Sources:
- pBR322 plasmid carrying V 2 type whole genomic DNA (designated pBR-V2): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Bower Gene International Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno-associated virus as a mammalian DNA cloning vector : transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81 : 6466-6470.
- the pCI-neo plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid pSG424 carrying the SV40 early promoter was purchased from Clonitic, USA.
- PSMA prostate-specific membrane antigen
- the recombinant adeno-associated virus vector carrying the prostate-specific membrane antigen (PSMA) gene or its mutant gene of the present invention is constructed by the following method (shown in Figure 1), and the specific process comprises the following steps:
- the nucleotide sequence containing the restriction enzymes EcoR I and EcoR V cleavage site (CGMTTCATGCGATATCGTT) was inserted into the plasmid
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and EcoR V, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions were: water bath at 4 ° C for 8 hours.
- the complete TR sequence at both ends is retained or the 75th nucleotide sequence of the TR at both ends of the AAV genome is inserted into a fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability of the r V virus and
- the method is as follows: First, the restriction endonuclease I (purchased from Promega, USA) is used to cut the TR at both ends, and the reaction system is: lwg The plasmid prepared above, 10U Ban I, 1. 5 l 10 X buffer Liquid G and 11.
- reaction system 500ng plasmid, 300ng 9 nucleotides Sequence, 10 IU T 4 DNA ligase
- CMV promoter SV40 early promoter by gene amplification technique (polymerase chain reaction, PCR).
- the specific method is as follows: Firstly, the CMV promoter is PCR-amplified under the guidance of primer 1 : AGATCTTCAATATTGGCCAT and primer 2: TGTCAGAAGCACTGACTGC with pCI-neo plasmid (purchased from Promega, USA).
- the PCR amplification conditions are as follows: 94 ° C 4% ⁇ After the end of the reaction, the PCR product was subjected to a 1.2% agarose gel.
- the reaction product was then centrifuged at a temperature of 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute, a total of 30 cycles; Electrophoresis detection, an expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain CMV.
- Promoter The SV40 early promoter was PCR-amplified under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC with PSG424 plasmid (purchased from Cloi tic, USA).
- the PCR amplification conditions were: 94 ° C for 4 minutes; Further, 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 40 seconds, a total of 30 cycles; the last 72 ° C for 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection An expected specific band appeared at 359 bp, and the target band was recovered and purified to obtain the SV40 early promoter.
- the PCR amplification conditions are as follows: 94 ° C for 4 minutes; then 94 ° C for 30 seconds 58 ° C 35 seconds, 72 ° C 1 minute 20 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, a 1176bp appeared The expected specific band is recovered and purified to obtain a beta actin promoter.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the PSMA cDNA (nt260-1512) was amplified by PCR under the guidance of primer 7: AGATGTGGAATCTCCTTCAC and primer 8: CMMTTGTTCTTCTAGGTC.
- the PCR amplification conditions were: 94 ° C 4 2 minutes agarose condensation on the PCR product after the end of the reaction.
- the reaction was completed at a temperature of 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute and 30 seconds for a total of 30 cycles; A desired specific band appeared at 1298 bp, and the target band was recovered and purified to obtain PSMA cDNA (nt260-1512).
- the cDNA fragment A was obtained by the same method as above (primer 7 and primer 9: CCACTGGGATTGAATTTTG, PSMA cDNA fragment B (primer 10: TTTCTTMACCGGACCT and primer 11: AATTTTCCCAGAGCAATTG) PSMA cDNA fragment C (primer 12: CATTAACGGTCTATACCCT and primer 13: ATAGTATCCAATTGGATG), PSMA cDNA fragment D (primer 15: CTACGTGTCTTCGAGGATC and primer 8), PSMA cDNA ( Ntl963-2514) (primer 16: CCAATGTTTAAATATCACCTC and primer 17: TTAGGCTACTTCACTTCAC), short PSMA cDNA fragment E
- primer 16 and primer 19 GAGTCTCTCACTGAACTTGG
- short PSMA cDNA fragment F primer 20: CAGGACTTTGACAAAAGCMC and primer 17
- the above expanded CMV promoter, SV40 early promoter, beta actin promoter, full-length PSMA cDNA or partial PSMA cDNA fragment are sequentially inserted into the modified pBR-AAV2 vector of step A,
- the first digestion reaction is carried out, and then the ligation reaction is carried out, wherein the digestion reaction system is: lyg plasmid; 10 U restriction enzyme I and 3 ⁇ 4 I (purchased from Promega, USA), 2. 5 ⁇ 1 10 X buffer C and 19.
- reaction conditions water bath at 37 ° C for 4 hours
- the reaction system is: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions were: water bath at 4 Torr for 8 hours.
- the plasmid carrying the promoter and the full-length PSMA cDNA were digested with restriction enzymes EcoR I and EcoR V, respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- the ligated DNA-rAAV/PSMA and rMV/mPSMA were introduced into genetically engineered Escherichia coli (F. colD DH5 a competent cells (Invitrogen, USA), and LB plates containing 10 ( ⁇ g/mL ampicillin) were used.
- E. colD DH5 a competent cells Invitrogen, USA
- LB plates containing 10 ⁇ g/mL ampicillin
- white single colonies were picked, plasmids were extracted and purified to obtain rAAV/PSMA plasmid and rAAV/mPSMA plasmid.
- Restriction endonuclease purchased from Promega, USA, followed by Nhe I EcoR V, Hind IWkEcoR I, Not IMhe I and 3 ⁇ 4 ⁇ I
- the restriction enzyme digestion was carried out, and the rMV vector without PSMA gene was used as a negative control (the CMV promoter, SV40 promoter and beta actin promoter were sequentially inserted into the modified pBR-MV2 vector of step A, and the vector was used for restriction.
- rAAV/PSMA Pst I endonuclease
- a 1298 bp specific band was obtained by Nhe I and EcoR V digestion, and a specific strip of 2.7 kb and 4.9 kb was obtained by digestion with f ⁇ I.
- the specific band of 1. lkb was obtained by digestion with 7 ⁇ 3 ⁇ 4 ⁇ ⁇ and ?e I, and specific bands of 0.37 kb and 1.8 kb were obtained by Pst I digestion, which was consistent with the expected results.
- the results of the digestion of the rMV/mPSMA plasmid were also consistent with the expected results.
- rAAV/PSMA plasmid and the rMV/mPSMA plasmid were further detected by gene amplification (PCR), wherein the detection result of the rAAV/PSMA plasmid is shown in Figure 2B (3. PCR gene amplification product), expanded An expected specific band of 1298 bp was obtained.
- PCR results of the rAAV/mPSMA plasmid were also consistent with the expected results (the size of the amplified product was rAAV/AmPSMA: 303 bp, rAAV/BmPSMA: 300 bp, rAAV/CmPSMA: 300 bp, rAAV/DraPSMA: 351 bp, and PSMA cDNA (ntl963).
- Example 2-2 Preparation of recombinant adeno-associated virus (rAAV) and determination of virus titer
- Example 2-1 Construction of recombinant adeno-associated virus vector rMV/PSMA carrying prostate specific membrane antigen (PSMA) gene and recombinant adeno-associated virus vector rMV/ carrying prostate specific membrane antigen (PSMA) mutant gene mPSMA (rAAV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA and rMV/DmPSMA).
- PSMA prostate specific membrane antigen
- PSMA prostate specific membrane antigen
- V-HEK293 cells containing adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the cell chromosome: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva) -Internati, M., Grizzi, F. Salati, E., Roman, JJ, Lim S., and Hermonat, PL Relax induction of cytotoxic T cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic Cells by an adeno- associated virus vector. Cancer Gene Therapy 8 : 948-957. ).
- adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the cell chromosome: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva) -Internati, M., Grizzi, F. Salati, E., Roman, JJ
- Liposomal transfection reagent Lipofectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 1.0 cm cell culture dish, for example, when V-HEK293 cells were grown in a carbon dioxide cell incubator to about a culture dish area. At 70%, do the following:
- Lipofectin l. O wg rAAV vector (rMV/PSMA or rAAV/mPSMA), 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50 ⁇ 0 ⁇ 1 with 10% The fetal bovine serum (or calf serum) was mixed in DMEM medium and allowed to stand at room temperature for 20 minutes.
- Prostate specificity of a gene carrying a tumor antigen The membrane antigen gene PSMA cDNA (nt260-1512) and its partial PSMA cDNA fragment (A, B, D, mutant gene) and the PSMA cDNA (ntl963-2514) and its partial fragments (E and F mutant genes) rAAV virus They were named rMV/PSMA, rAAV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA> rAAV/DmPSMA and rMV/PSMA cDNA (ntl963-2514), rAAV/EmPSMA and rMV/FmPSMA.
- the various r V viruses obtained in step 1 using conventional dot blot hybridization (r V/PSMA, rAAV/AmPSMA ⁇ rAAV/BmPSMA rAAV/CmPSMA.rAAV/DmPSMA and rAAV/PSMA cDNA (ntl963-2514), rAAV/ EmPSMA and rAAV/FmPSMA)
- the specific method includes the following steps: Only the DNA probe used is a specific probe for the PSMA gene.
- RMV virion DNA was extracted using conventional DNA phenol/chloroform extraction.
- the nylon membrane was placed in a dot blotter, and the alkali-denatured rAAV virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- the probe is "PSMA cDNA (nt260-1512) or PSMA cDNA obtained in Step 2-1 of Example 2-1 (ntl963- 2514)
- the PCR amplification products were subjected to 1.2% agarose gel electrophoresis, and the PCR amplification products were detected under ultraviolet light. The results showed that positive bands appeared, indicating that the probe was successfully labeled.
- rMV/PSMA the detection result of rMV/PSMA is shown in Fig. 4B, and the virus titer of rAAV/PSMA is 10 1D - 10 11 copies/mL.
- the virus titer of rAAV/PSMA cDNA (ntl963-2514) and the six rMV/mPSMA can also reach 10 111 - 10 12 copies/mL.
- Example 2-3 tumor antigen-inducing monocyte-macrophage-dendritic cell line killing tumor experimental materials and sources thereof:
- rAAV virus rAAV/PSMA, rAAV/PSMA cDNA (ntl963-2514) and rAAV/mPSMA (rAAV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA, rAAV/DmPSMA, rAAV/EmPSMA, rAAV/FmPSMA).
- AB1-V Cell Culture Medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the entire process of killing a tumor-based tumor cell-infected tumor of a tumor carrying a prostate cancer-specific membrane antigen PSMA gene and a mutant gene thereof comprises the following steps:
- peripheral blood mononuclear cells were obtained by the method, mixed with the AB1-V medium, added to the cell culture flask, and cultured in a constant temperature carbon dioxide incubator for 2 hours.
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- Example 2-2 of the present invention Adding one (or more, better effect)
- the rAAV virus obtained in Example 2-2 of the present invention is added in an amount of about 100-1000 MOI, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- a specific fluorescent antibody against the tumor antigen-prostate-specific membrane antigen PSMA (purchased from R&D, USA) was used to label the mononuclear cells infected with rAAV of the present invention or immature. DC, and then flow cytometry to detect the number of positive cells.
- the efficiency of detection of peripheral blood mononuclear cells by recombinant adeno-associated virus rAAV/PSMA is shown in Fig. 6B, and the efficiency of VrMV/PSMA infection of peripheral blood mononuclear cells is 92%, and various tumor-bearing antigens are constructed and prepared.
- r V ( rAAV/AmPSMA , rAAV/BmPSMA , rAAV/CmPSMA , rAAV/DraPSMA , rAAV/PSMA cDNA (nt 1963-2514 ) , rAAV/EmPSMA , rAAV/FmPSMA ) are all about the efficiency of infection of peripheral blood mononuclear cells 80% - 90%, demonstrating that the rAAV of the present invention has a high infection efficiency.
- DC dendritic cells
- DC expression levels of CD80, CD83, and CD86 are positively correlated with DC function.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- the DC is a control.
- CD80, CD83 and CD86 in DCs of recombinant adeno-associated virus rAAV/PSMA infection is shown in Figure 7B, rAAV/PSMA and other rAAVs carrying tumor antigens (rAAV/AmPSMA, rAAV/BmPSMA, rAAV/ CmPSMA, rAAV/DmPSMA, rAAV/PSMA cDNA (nt 1963-2514), rAAV/EmPSMA, rAAV/FmPSMA)
- the level of CD molecules expressed by infected DCs was significantly higher than that of controls, demonstrating the construction and preparation of tumor-bearing antigen-prostate specific After induction of peripheral blood mononuclear cells by the membrane antigen PSMA and its mutant r V, the induced DC is powerful.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells are positively correlated with the expression level of IFN- ⁇ .
- the level of CTL expressing IFN-y induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (without stimulation) .
- the 22" DC-induced CTL was used as a control.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescent marker against IFN- ⁇ .
- the antibody purchased from BD was finally detected by flow cytometry.
- the IFN- y expression level of CTL induced by DC infected with r V/PSMA is shown in Fig. 8B. It is carried by rAAV/PSMA and others.
- rAAV/AmPSMA Tumor antigens of rAAV (rAAV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA, rAAV/DmPSMA, rAAV/PSMA cDNA (ntl963-2514), rAAV/EmPSMA.
- rAAV/FmPSMA induced CTL expression of IFN- ⁇ The level of CTL was significantly higher than that of the control, which demonstrated that the tumor-bearing antigen-prostate-specific membrane antigen and its mutant r-infected DC constructed and prepared by the present invention were powerful.
- CTL Cytotoxicity T lymphocyte
- step one was subjected to rAAV (rAAV/PSMA.rAAV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA, rAAV/DmPSMA, rAAV/PSMA cDNA (nt 1963-2514), rAAV/EmPSMA, rAAV/FmPSMA
- DC-induced cytotoxic T lymphocytes are mixed with prostate cancer cells by 20:1 (lymphocytes: tumor cells), and CTL killing tumors are detected by conventional sputum method and 51 Cr (chromium-51) killing test. Cell activity.
- Fig. 9B The statistical results of tumor cell killing rate of CTL induced by rAAV/PSMA-infected DC are shown in Fig. 9B, and the DC antigen-bearing membrane antigen and its mutant rAAV-infected DC constructed and prepared by the present invention are constructed and prepared by the present invention.
- the induced CTL can lyse (kill) tumor cells more effectively, and the killing rate can reach about 50%.
- Prostate cancer cells were isolated from three patients with prostate cancer (A, B, C). The lung, pancreas, liver, and kidney cells were used as controls. The same method was used to detect rAAV (rAAV/PSMA, rAAV) in step 1. /AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA, rAAV/DmPSMA, rAAV/PSMA cDNA (ntl963-2514),
- rAAV/EmPSMA, rAAV/FmPSMA Cytotoxicity induced by infected DCs Specificity of T lymphocytes killing tumor cells.
- the tumor cell killing specific detection result of the CTL induced by the DCA/PSMA-infected DC is as shown in FIG. 9B, and the tumor-bearing antigen-prostate-specific membrane antigen and its mutant r V constructed and prepared by the present invention are prepared and prepared.
- the CTL induced by the infected DC has no killing effect on the above cells, and it is proved that the CTL induced by the tumor-bearing antigen-prostate-specific membrane antigen and its mutant rAAV-infected DC constructed and prepared by the present invention has antigen specificity, that is, No killing effect on antigen-negative cells.
- rAAV-DC tumor antigen-prostate-specific membrane antigen PSMA and its mutant rAAV-infected DC
- the rV rAAV/PSMA, rMV/AmPSMA, rAAV/BmPSMA, rAAV/CmPSMA, rAAV/DmPSMA, rAAV/PSMA cDNA of Example 2-3
- Nt 1963-2514 rAAV/EmPSMA and rAAV/FmPSMA
- rAAV-DC infected DCs
- Treatment Cheng Usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to 1 to 3 months.
- Therapeutic effect reaction after rAAV-DC treatment
- the statistical results are shown in Table 2-1 (B: serum tumor markers decreased or disappeared.
- Q The patient's quality of life improved. If the pain is reduced or disappeared, the appetite is increased, etc.
- C CT or PET-CT shows that the cancer lesions or metastatic lesions are significantly reduced or disappeared.)
- Adverse reactions In most cases, mild flu-like reactions occur within a short period of time after treatment, but the patients can withstand, and the symptoms disappear in a short period of time.
- rMV-DC CTL induced by the rAAV-infected DC of the present invention
- rAAV-DC Recombinant adeno-associated virus-dendritic cell technology
- the imaging changes of the metastatic lesions before and after treatment in 14 patients with prostate cancer in step 1 were performed.
- the imaging findings of a change of metastatic lesions before and after CTL treatment induced by rMV/PSMA-infected DCs in stage IV are shown in Fig. 10B.
- the results showed that the metastatic lesion of the patient disappeared significantly after the CTL treatment induced by the r V-infected DC (rMV-DC) of the present invention, further demonstrating that it was infected with the rAAV of the present invention.
- the CTL induced by DC can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients, and can be used for preparing antitumor drugs.
- the changes of serum tumor marker-tumor-associated antigen PSA levels were detected. Four of them were treated with rMV/PSMA-infected DC before and after CTL treatment.
- the changes in PSMA tumor-associated antigen levels in the serum of patients are shown in Figure 11B, and the results were obtained by the present invention rAAV (rMV/PSMA, rAAV/AmPSMA, rAAV/BmPSMA, rAAV/mPSMA, rAAV/DmPSMA rAAV/PSMA cDNA (nt 1963- 2514), rAAV/EmPSMA and rMV/FmPSMA), one or both of the infected DCs (rAAV-DC) induced CTL treatment, the serum levels of tumor antigen PSMA were mostly decreased, indicating that the tumor in vivo The load was significantly reduced (the tumor cells were significantly reduced), further demonstrating that the CTL induced by the DC infected with the rMV
- Example 3-1 Construction and detection of recombinant adeno-associated virus vector r V/CEA and rAAV/mCEA Materials and sources:
- A. pBR322 plasmid carrying AAV type 2 whole genome DNA (designated pBR-V2): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81 : 6466-6470.
- the pCI-ne plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid pSG424 carrying the SV40 early promoter was purchased from Clonitic, USA.
- CEA chorioembryonic antigen
- reaction system Reconstruction of the pBR-AAV2 plasmid by the restriction enzymes ⁇ and J3 ⁇ 43 ⁇ 4 I (purchased from Promega, USA), the structural gene Rep of the adeno-associated virus V genome in the pBR-AVV2 plasmid / / (3 ⁇ 4o gene completely excised, the reaction system is: lg pBR-MV2, 10U Bst98 I, 10U Hpa I, 2.5 l 10X buffer D and 19.5 ⁇ 1 deionized water; reaction conditions are: water bath at 37 ° C 4 Then, the nucleotide sequence (CGMTTCATGCGATATCGTT) containing the restriction enzymes EcoR I and EcoR ⁇ cleavage site was inserted into the plasmid, and the reaction system was: 500 ng plasmid, 300 ng of the nucleotide sequence of EcoR I and EcoR, 10 IU of T 4 DNA ligase (purchased from Promega, USA), 1.5 ⁇ l of 10
- the sequence or the 75th nucleotide sequence of the TR at both ends of the AAV genome is inserted into a fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability of the r V virus and improve the replication efficiency of the virus by : First use within the restrictions
- the enzyme I purchased from Promega, USA
- reaction conditions were: water bath at 4 ° C for 8 hours.
- Amplification of the CMV promoter, SV40 early promoter by gene amplification technology (polymerase chain reaction, PCR).
- the specific method is as follows: Firstly, the CMV promoter was PCR-amplified under the guidance of primer 1: AGATCTTCAATATTGGCCAT and primer 2: TGTCAGAAGCACTGACTGC with pCI- neo plasmid (purchased from Promega, USA).
- the PCR amplification conditions were as follows: 94 ° C 4 minutes; further 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute, a total of 30 cycles; the last 72 ° C for 8 minutes, after the reaction, the PCR product was tested by 1.2% agarose gel electrophoresis An expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ⁇ 40, seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, a 359bp appeared The expected specific band was recovered and purified to obtain the SV40 early promoter.
- the recombinant adeno-associated virus vector of the present invention is specifically prepared by separating nucleic acid and raRNA from human primary colon cancer cells by using nucleic acid separation technology (also If the total DNA and m RNA are obtained synthetically or commercially, and then using the total DNA as a template, PCR-amplification of the beta actin promoter under the guidance of primer 5: CCCGGGCCCAGCACCCCAAG and primer 6: CATCCATGGTGAGCTGCG, PCR amplification conditions For: 94 first.
- the PCR product was 1.2% agar A desired specific band appeared at 1176 bp by glycogel electrophoresis, and the band of interest was recovered and purified to obtain a beta actin promoter.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the full-length CEA cDNA was amplified by PCR under the guidance of primer 7: CATGGAGTCTCCCTCGGC and primer 8: TGCTATATCAGAGCGCAACC.
- CEA cDNA fragment A (primer 7 and primer 9: AGTGAGGGTCCTGTTGCCATTG) and CEA cDNA fragment were obtained by the same method as above.
- B (primer 10: CTATTCAATGTCACAAGAAATG and primer 11: ATAGAGGACATTCAGGATG)
- CEA cDNA fragment C (primer 12: GGCCCAGACGACCCCAC and primer 8).
- the above expanded CMV promoter, SV40 early promoter, beta actin promoter, full-length CEA cDNA or partial CEA cDNA fragment are sequentially inserted into the modified pBR-AVV2 vector of step A,
- the first digestion reaction is carried out, and then the ligation reaction is carried out, wherein the digestion reaction system is: lg plasmid; 10 U restriction endonuclease ⁇ rf l and I (purchased from Promega, USA), 2. 5 ul 10 X buffer C and 19.
- reaction conditions water bath at 37 ° C for 4 hours
- the reaction system is: 500 ng of the plasmid after digestion, 300 ng of promoter DNA, 10 IU of T 4 DNA Enzyme (purchased from Promega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions were: water bath at 4 ° C for 8 hours.
- the plasmid carrying the promoter and the full-length PSA cDNA were digested with restriction enzymes EcoR I and EcoR V, respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length CEA cDNA (designated rAAV/CEA), and the CMV promoter carrying the SV40 early promoter were obtained.
- ⁇ -actin promoter and A or B or C different CEA cDNA fragments (mutant) of recombinant adeno-associated virus vectors named rAAV/AmCEA, rAAV/BmCEA and r V/CmCEA, respectively, collectively named rMV/mCEA ).
- the ligated DNA-rAAV/CEA and rAAV/mCEA were introduced into genetically engineered Escherichia coli (£ coli) DH5 a competent cells (Invitrogen, USA), respectively, using LB plates containing 10 ( ⁇ g/mL ampicillin). For resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain r V/CEA plasmid and rMV/mCEA plasmid.
- the purified rAAV/CEA plasmid and the rAAV/mCEA plasmid obtained in the first step were digested with a restriction enzyme (only, % EcoR MamH 1 , Not 1, BamH IMsi I ), and the restriction endonuclease was used. Enzymes are purchased from the United States Country Promega.
- the rAAV vector without CEA gene was used as a negative control (the CMV promoter, SV40 early promoter, and beta actin promoter were sequentially inserted into the modified PBR-MV2 vector of step A, and the vector was replaced with restriction endonuclease.
- rAAV/CEA ( ⁇ rf I and Nsi I endonuclease). And a specific band of 2 bp was obtained by digestion with EcoR I and BamH I. A specific band of 4 kb was obtained by digestion with Not I, and 1. 3 kb and 2. were obtained by digestion with BamH I and Nsi I. The specific band of lkb is consistent with the expected results. The results of the digestion of the rAAV/mCEA plasmid were also consistent with the expected results.
- rMV/CEA plasmid and the rAAV/mCEA plasmid were further tested by gene amplification (PCR), and the results of the rMV/CEA plasmid were shown in the right panel of Figure 2C (recombinant adeno-associated virus vector rAAV/CEA).
- PCR detection results 1. DNA molecular weight standard 2. Negative control 3. PCR amplification product of CEA cDNA. ), 2115 bp of the expected specific band was obtained by amplification.
- the PCR results of the rAAV/mCEA plasmid were also consistent with the expected results (the size of the amplified product was r V/AmPSA: 603 bp rAAV/BmPSA: 636 bp, rAAV/CmPSA: 870 bp).
- the above detection results indicated that the recombinant adeno-associated virus vector rAAV/CEA carrying the carcinoembryonic antigen (CEA) gene and the recombinant adeno-associated virus vector carrying the carcinoembryonic antigen (CEA) mutant gene were obtained with the correct insertion position and sequence. /mCEA.
- Example 3-2 Preparation of recombinant adeno-associated virus (rMV) and determination of virus titer
- Example 3-1 Construction of recombinant adeno-associated virus vector carrying carcinoembryonic antigen (CEA) gene rAAV/CEA and recombinant adeno-associated virus vector carrying carcinoembryonic antigen (CEA) mutant gene rAAV/mCEA
- AAV-HEK293 cells containing adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the chromosome of the cell: established by the Gene Therapy Center of the affiliated Hospital of the University of Arkansas. (Liu, Y., Chiriva-Internati, M., Grizzi, F. Salati, E., Roman, JJ, Lim S., and Hermonat, PL Rapid induction of cytotoxic T cell response against cervical cancer cel ls by human papi llomavirus Type 16 E6 antigen gene del ivery into human dendrit ic cells by an adeno- associated virus vector. Cancer Gene Therapy 8 : 948 - 957. ).
- D. Liposomal transfection reagent Lipofect in purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 1.0 cm cell culture dish, for example, when V-HEK293 cells were grown in a carbon dioxide cell incubator to about a culture dish area. At 70%, do the following:
- A. follow the instructions for Lipofectin: l. O wg rMV vector (rMV/CEA or rAAV/mCEA), 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50. 0 ⁇ 1 with 10% The fetal bovine serum (or calf serum) was mixed in DMEM medium and allowed to stand at room temperature for 20 minutes.
- rAAV/CEA rAAV/AmCEA
- rMV/BmCEA rAAV/CmCEA
- the virus titer was determined by routine dot blot hybridization of various rMV viruses (rAAV/CEA, rMV/AmCEA, rAAV/BmCEA, rAAV/QnCEA) obtained in step 1.
- the specific method includes the following steps: only the DNA used The needle is a specific probe for the tumor antigen gene.
- the nylon membrane was placed in a dot blotter, and the alkali-denatured rAAV virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- Example 3-1 Step C.
- the PCR amplification products were subjected to 1. 2% agarose gel electrophoresis, and the PCR amplification products were detected under ultraviolet light. The results showed positive bands, indicating that the probes were successfully labeled.
- the detection results of rAAV/CEA are shown in Fig. 4C, the virus titer of rAAV/CEA is 10' 2 copies/mL, and the virus titer of rAAV/mCEA is 10"-10 12 copies/mL.
- Example 3-3 tumor antigen-inducing monocyte-macrophage-dendritic cell line killing tumor experimental materials and sources thereof:
- AIM-V Cell Culture Medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the whole process of killing tumors based on the monocyte-based tumor of a tumor patient infected with one or more rAAV viruses carrying a tumor antigen gene includes The following steps:
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- Example 3-2 of the present invention Adding one (or more, better effect)
- the rAAV virus obtained in Example 3-2 of the present invention is added in an amount of about 100-1000 MOI, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- the old medium was removed, and the AIM-V medium containing GM-CSF, IL-4 (800 IU/mL) and TNF-a (20 IU/mL) was added, and the culture was continued.
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- the mononuclear cells infected with the rAAV of the present invention obtained by labeling the first step against the tumor antigen-carcinoembryonic antigen CEA and its mutant specific fluorescent antibody (purchased from BD, USA) Mature DC, and then flow cytometry to detect the number of positive cells.
- the efficiencies of the recombinant adeno-associated virus rAAV/CEA infection of peripheral blood mononuclear cells are shown in Fig. 6C, and the efficiency of rAAV/CEA infection of peripheral blood mononuclear cells is 92.8%, and various carriers are constructed and prepared.
- the tumor antigen r V (r V / AmCEA, rAAV / BmCEA. rAAV / CmCEA) infection of peripheral blood mononuclear cells are about 90%, that is, about 90% of peripheral blood mononuclear cells can be r V Viral infection demonstrated that the rAAV of the present invention has a high infection efficiency.
- DC dendritic cells
- the levels of DC expression CD80, CD83 and CD86 were positively correlated with the function of DC.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- the DC is a control.
- CD80, CD83 and CD86 in DCs of recombinant adeno-associated virus rMV/CEA infection is shown in Figure 7C, rAAV/CEA and other rAAVs carrying tumor antigens (rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA)
- rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA The level of CD molecules expressed by infected DCs was significantly higher than that of the control, demonstrating the construction and preparation of DCs carrying tumor antigen-carcinoembryonic antigen CEA and its mutant rAAV after infection of peripheral blood mononuclear cells. Powerful.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN-y induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry.
- the IFN- ⁇ expression level of CTL induced by DCs infected with rAAV/CEA is shown in Fig. 8C, and is rV/CEA and other rAAVs carrying tumor antigens (rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA).
- the level of CTL expression of IFN- ⁇ induced by infected DC was significantly higher than that of the control, demonstrating that CTLs induced by the tumor antigen-bearing CEA-carcinoembryonic antigen and its mutant rAAV-infected DC constructed and prepared by the present invention are powerful.
- CTL Cytotoxicity T lymphocyte
- the cytotoxic T lymphocytes induced by DCs infected with r V were respectively 20:1 (lymphocytes: tumor cells)
- the activity of CTLs to kill tumor cells was examined by conventional sputum method and 5l Cr (chromium-51) killing test.
- the statistical results of tumor cell killing rate of CTLs induced by DCs infected with rAAV/CEA are shown in Fig. 9C, and are induced by DCs carrying tumor antigen-carcinoembryonic antigen and mutant rAAV thereof constructed and prepared by the present invention.
- CTL can lyse (kill) tumor cells more effectively, with a killing rate of up to 50%.
- the CEA antigen-negative stomach, lung, intestine, and breast cells were used as controls, and the same method as above was used to detect rAAV (rAAV/CEA > rAAV/AmCEA, rAAV/BmCEA, rAAV/CmCEA)
- the specificity of cytotoxic T lymphocytes induced by infected DCs to kill tumor cells is as shown in FIG. 9C, and the rV infection carrying the tumor antigen-carcinoembryonic antigen CEA and its mutant form constructed and prepared by the present invention is shown.
- the CTL induced by DC has no killing effect on the above CEA antigen-negative cells, and it is proved that the CTL induced by the tumor antigen-carcinoembryonic antigen CEA and its mutant rAAV-infected DC constructed and prepared by the present invention has antigen specificity, That is, there is no killing effect on antigen-negative cells.
- rAAV-DC tumor antigen-carcinoembryonic antigen CEA and its mutant r V-infected DC
- adeno-associated virus-dendritic cell technology which will be the invention of rAAV in Example 3-3
- One or two infected DCs rAAV/CEA, rAAV/AmCEA, rAAV/BmCEA and rAAV/CmCEA
- the infusion volume is IX 10 9 -5 X 10 9 .
- Treatment course usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to once every 1-3 months. To test its anti-tumor effect in vivo.
- rAAV-DC CTL induced by the rAAV-infected DC
- the imaging changes of 20 cases of breast cancer, colon cancer, gastric cancer and lung adenocarcinoma in the first step before and after treatment were performed.
- One case of stage IV metastatic colon cancer was induced by rAAV/CEA-infected DC.
- the results of imaging observations of changes in metastatic lesions before and after treatment are shown in Fig. 10C (left picture is before treatment, right picture is after four months of treatment), and the result is r V(r V/CEA, r V/ according to the present invention.
- the metastatic lesion of the patient disappeared significantly, further demonstrating that the DC infected by the rAAV of the present invention
- the induced CTL can effectively inhibit the growth of malignant tumor cells or kill tumor cells in a patient, and can be used for preparing an antitumor drug.
- Example 4-1 Construction and detection of recombinant adeno-associated virus vector r V/K19 and r V/mK19 Materials and sources:
- A. pBR322 plasmid carrying MV type 2 whole genome DNA (designated pBR-V2): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81: 6466-6470.
- nasopharyngeal carcinoma cells Isolated from cancer tissues of patients with nasopharyngeal carcinoma, immunohistochemistry confirmed keratin 19 antigen (K19 or CK19) positive; or obtained commercially.
- the pCI-ne plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid pSG424 carrying the SV40 early promoter was purchased from Clonitic, USA.
- the recombinant adeno-associated virus vector carrying the keratin 19 (cytokeratin 19, K19 or CK19) gene or its mutant gene of the present invention is constructed by the following method (as shown in Fig. 1), and the specific procedure includes the following steps:
- the 3 ⁇ 4 ⁇ gene was completely excised, and the reaction system was: lwg pBR-AAV2, 10U Bst98 I, 10U Hpa I, 2: 5 ⁇ 1 10X buffer D and 19.5 ⁇ l of deionized water; the reaction conditions were: water bath at 37 Torr for 4 hours.
- the nucleotide sequence containing the restriction enzymes EcoR I and EcoR V cleavage sites was inserted into the plasmid.
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and EcoR V, 10 IU.
- T 4 DNA ligase purchased from Promega, USA
- 1.5 ⁇ l 10 XT 4 DNA ligation buffer and 11.5 ⁇ l of deionized water reaction conditions were: water bath at 4 ° C for 8 hours.
- the reaction system is: lug The plasmid prepared above, 10U Ban I, 1.5 ul 10X buffer G and 11.5 ⁇ l deionized water; reaction conditions: water bath at 37 ° C for 4 hours, then insert 9 nucleotide fragments Into the plasmid, the reaction system is: 500 ng plasmid, 300 ng 9 nucleotide sequence, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ 1 deionized water; reaction conditions are: water bath at 4
- Amplification of the CMV promoter, SV40 early promoter by gene amplification technology (polymerase chain reaction, PCR).
- the specific method is as follows: Firstly, the CMV promoter is PCR-amplified under the guidance of primer 1 : AGATCTTCAATATTGGCCAT and primer 2: TGTCAGAAGCACTGACTGC with pCI-neo plasmid (purchased from Promega, USA).
- the PCR amplification conditions are as follows: 94 ° C 4 minutes; further 94 ° C 30 seconds, 60 ° C 35 seconds, 72 ° C 1 minute, a total of 30 cycles; finally 72 ° C 8 minutes, after the reaction, the PCR product was 1.2% agarose gel After electrophoresis detection, an expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ° C 40 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, An expected specific band appeared at 359 bp, and the target band was recovered and purified to obtain the SV40 early promoter.
- K19 cDNA and partial K19 cDNA fragments (named A (from the 5'-end 50-895 base), B (from 5, end 481-895) (base base), (: (from the 5' end, 4812-1265 bases), in this example, the above fragment is exemplified but not limited to the above fragment, and other K19 cDNA fragments having the same function as the K19 cDNA can be used for construction.
- the recombinant adeno-associated virus vector of the present invention is specifically prepared by separating nucleic acid and mRNA from human primary prostate cancer cells by using nucleic acid separation technology (the total DNA and mRNA can also be artificially synthesized or commercially obtained), and then Using DNA as a template, the beta actin promoter was PCR-amplified under the guidance of primer 5: CCCGGGCCCAGCACCCCAAG and primer 6: CATCCATGGTGAGCTGCG. The PCR amplification conditions were: 94°C for 4 minutes; then 94°C for 30 seconds, 58°C.
- PCR product was detected by 1.2% agarose gel electrophoresis, an expected specific at 1176 bp Sex strips, The target band was recovered and purified to obtain the beta actin promoter.
- the mRNA was reverse transcribed into its cDNA and used as a template to PCR-amplify full-length K19 under the guidance of primer 7: CTCGCCATGACTTCCTACAG and primer 8: TGCTGCCTCAGAGGACCTTG.
- PCR amplification conditions are: first 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 59 ° C for 35 seconds, 72 ° C 1 minute 30 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, the reaction is over Then, the PCR product was subjected to 1.2% agarose gel electrophoresis, and a desired specific band appeared at 1216 bp. The desired band was recovered and purified to obtain a full-length K19 cDNA, and K19 was obtained by the same method as above.
- cDNA fragment A (primer 7 and primer 9: CCGGTTCAATTCTTCAGTCC), K19 cDNA fragment B (primer 10: TGGTGCCACCATTGAGAACT and primer 9), K19 cDNA fragment C (primer 10 and primer 8).
- reaction conditions were: water bath at 37 ° C for 4 hours
- the reaction system was: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU of T 4 DNA ligase ( Purchased from Promega, USA, 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions were: water bath at 4 ° C for 8 hours.
- the plasmid carrying the promoter and the full-length PSA cDNA were digested with restriction enzymes EcoR I and EcoR V, respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length K19 cDNA (designated r V/K19 ) was obtained, and the CMV promoter and the SV40 promoter were carried.
- beta actin promoter and A or B or C different K19 cDNA fragments (mutant) recombinant adeno-associated virus vectors (named r V/AmK19, rAAV/BniK19 and r V/CmK19, respectively, named rAAV/ mK19).
- the ligated DNA-rAAV/K19 and r V/mK19 were introduced into the genetically engineered E. coli coli) DH5 a competent cells (Invitrogen, USA), using LB plates containing 10 ( ⁇ g/mL ampicillin). For resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain rAAV/K19 plasmid and rAAV/mK19 plasmid.
- the purified r V/K19 plasmid and the rAAV/mK19 plasmid obtained in the first step were subjected to restriction enzymes (sequence, EcoR Ba H 1 , EcoR NkNot I, EcoR NkSal I, EcoR EcoR 1 , Pstl ).
- restriction enzymes used were all purchased from Promega, USA.
- the digested product was detected by 1. 2% agarose gel electrophoresis, and the detection result of rAAV/K19 plasmid is shown in the left figure of Fig. 2D.
- Example 4-1 Construction of recombinant adeno-associated virus vector carrying keratin-19 (K19) gene rMV/K19 and recombinant adeno-associated virus vector carrying keratin-19 (K19) mutant gene r V/mK19
- Liposomal transfection reagent Lipofectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 1.0 cm cell culture dish, and the AAV-HEK293 cells were grown in a carbon dioxide cell incubator to an area of the culture dish. At 70%, do the following:
- Lipofectin l. O g rAAV vector (rMV/K19 or rAAV/mK19), 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50. 0 ⁇ 1 with 10% The fetal bovine serum (or calf serum) was mixed in DMEM medium and allowed to stand at room temperature for 20 minutes.
- rAAV viruses carrying the full-length K19 cDNA of the tumor-associated antigen gene keratin-19 gene or a partial K19 cDNA fragment were named rAAV/K19, rAAV/AmK19, rAAV/BmK19, respectively. rAAV/CmK19.
- the virus titer was determined by the conventional dot blot hybridization method for various rAAV viruses (rAAV/K19, rMV/AmK19, rAAV/BmK19, r V/CmK19) obtained in the first step, and the specific method includes the following steps: only the DNA used
- the probe is a specific probe for a tumor associated antigen gene.
- the rAAV virus particle DNA was extracted using a conventional DNA phenol/chloroform extraction method.
- D Prepare a DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is the CK19 cDNA obtained in Example 4-1, Step C. After PCR amplification, PCR amplification The product was subjected to 1.2% agarose gel electrophoresis, and the PCR amplification product was detected under ultraviolet light, and a positive band appeared, indicating that the probe was successfully labeled.
- the detection result of rMV/K19 is shown in Fig. 4C
- the virus titer of rAAV/K19 is 10 12 copies/mL
- the virus titer of three rMV/mkl9 can also reach ⁇ - ⁇ 12 copies/mL.
- Example 4-3 Tumor-associated antigen K19 gene introduction into monocyte-macrophage-dendritic cell line killing tumor experiment
- rAAV virus rMV/K19 and rMV/mK19 (rMV/AmK19, rAAV/BmK19.rAAV/CmK19).
- AIM-V cell culture medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF-a) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF-a tumor necrosis factor
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- Example 4-2 of the present invention Adding one (or more, better effect)
- the rAAV virus obtained in Example 4-2 of the present invention is added in an amount of about 100-1000 M0I, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- the mononuclear cells infected with the rAAV of the present invention obtained by labeling step one with a specific fluorescent antibody (purchased from BD, USA) against the tumor-associated antigen-keratin-19 and its mutant Immature DCs were then flow cytometry to detect the number of positive cells.
- a specific fluorescent antibody purchased from BD, USA
- the efficiency of detecting the peripheral blood mononuclear cells of the recombinant adeno-associated virus rAAV/K19 is shown in Fig. 6D, and the efficiency of rAAV/K19 infection of peripheral blood mononuclear cells is 94.3 %, and various carriers are constructed and prepared.
- Tumor-associated antigen rAAV (r V/AmK19, rAAV/BmK19, rAAV/CmK19) is about 90% efficient in infecting peripheral blood mononuclear cells, ie about 90% of peripheral blood mononuclear cells can be r V Viral infection, demonstrating that the r V of the present invention has a high infection efficiency.
- DC dendritic cells
- DC expression levels of CD80, CD83, and CD86 are positively correlated with DC function.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- the DC is a control.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN- ⁇ induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry.
- rAAV/K19 the expression level of IFN- ⁇ of CTL induced by DC infected with rAAV/K19 is shown in Fig. 8D, and rAAV/K19 and other rAAVs carrying tumor-associated antigens (rAAV/AmK19, rAAV/BmK19, rAAV/CraK19)
- the level of IFN- ⁇ expressed by infected DCs was significantly higher than that of the control, demonstrating that CTLs induced by the tumor-associated antigen-keratin-19 and its mutant rMV-infected DCs constructed and prepared by the present invention are powerful. .
- CTL Cytotoxicity T lymphocyte
- the rAAV (rAAV/K19, rAAV/AmK19, rMV/BmK19, rAAV/CmK19) Infected DC-induced cytotoxic T lymphocytes are mixed with K19-positive primary nasopharyngeal carcinoma cells, cervical cancer and lung squamous cell carcinoma according to 20:1 (lymphocytes: tumor cells), using traditional The ⁇ method and the 51 Cr (chromium-51) killing test were used to detect the activity of CTLs to kill tumor cells.
- the statistical results of tumor cell killing rate of CTL induced by rAAV/K19-infected DC are shown in Fig.
- CTL induced by non-stimulated DC can lyse (kill) tumor cells more effectively, with a kill rate of more than 50%.
- the CTL induced by DC has no killing effect on K19 antigen-negative cancer cells, and it is proved that the CTL induced by the tumor-associated antigen-keratin-19 and its mutant rAAV-infected DC constructed and prepared by the present invention has antigen specificity. That is, there is no killing effect on antigen-negative cells.
- rMV-DC tumor-associated antigen-keratin 19 and its mutant-infected DC
- Recombinant adeno-associated virus-dendritic cell technology which is the DC of Example 4-3 infected with one or both of the rAAVs of the present invention (rAAV/K19, rAAV/AmK19, rAAV/BmK19 and rMV/CmK19) (rAAV-DC) induced CTL reinfusion of 15 patients with cervical cancer, lung squamous cell carcinoma and nasopharyngeal carcinoma, the infusion volume was 1 X 10 9 - 5 X 10 9 CTL cells.
- Treatment course Usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1 - 2 times a month, and further reduced to once every 1-3 months.
- Therapeutic effect (reaction after rAAV-DC treatment) The statistical results are shown in Table 4-1 (B: serum tumor markers decreased or disappeared. Q: The patient's quality of life improved. If the pain is reduced or disappeared, the appetite is increased, etc. C : CT or PET-CT shows a marked reduction or disappearance of cancer lesions or metastatic lesions.), Adverse reactions: In most cases, mild flu-like reactions occur within a short period of time after treatment, but patients can withstand, and the symptoms disappear in a short period of time. No serious adverse reactions and toxic reactions were observed.
- the changes of serum tumor marker-tumor-associated antigen CK19 levels were detected. Four of them were infected with rAAV/K19.
- the changes of serum CK19 tumor-associated antigen levels in patients with lung squamous cell carcinoma before and after CTL treatment induced by DC are shown in Fig. 11D, and the results are in the rAAV (rAAV/K19, rMV/AmK19, rAAV/BmK19 and rAAV/CmK19) of the present invention.
- the level of tumor-associated antigen CK19 in the serum decreased significantly, indicating that the tumor burden in the patient was significantly reduced (the tumor cells were significantly reduced), further proof
- the CTL induced by the DC-infected DC of the present invention can effectively inhibit the growth of malignant tumor cells or kill tumor cells in a patient, and can be used for preparing an antitumor drug.
- Part V Recombinant adeno-associated virus vector rAAV/ Her-2/neu
- Example 5-1 Construction and detection of recombinant adeno-associated virus vectors rAAV/Her- 2/neu and rAAV/mHer-2/neu
- A. pBR322 plasmid carrying AAV type 2 whole genome DNA (designated pBR-AAV2): prepared by Professor Paul L. Hermonat, one of the principal technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector : transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81 : 6466-6470.
- Human breast cancer cells isolated from cancer tissues of breast cancer patients, immunohistochemically confirmed Her-2/neu antigen positive or commercially available.
- the pCI-ne plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid carrying the SV40 early promoter, PSG424, was purchased from Clonitic, USA.
- Amplification nucleotide primers The company's mRNA sequence of human Her-2/neu antigen gene published in the US gene bank (US NCI gene library: NM_004448), its gene length is 3768bp design pair expansion Primers that increase the full-length Her-2/neu cDNA, based on the outer envelope (encoding amino acid residues 153-653 from the amino terminus), intermediate region (encoding amino acid residues 403-906 from the amino terminus) As well as the gene sequence of the inner region of the envelope (encoding amino acid residues from amino terminus at positions 79-12555), three pairs of primers were designed to obtain the three mutant genes, respectively.
- the recombinant adeno-associated virus vector carrying the Her-2/neu antigen gene or its mutant gene of the present invention is constructed by the following method (as shown in Fig. 1), and the specific process comprises the following steps: I. Construction of recombinant adeno-associated virus vector
- reaction system Reconstruction of pBR-AAV2 plasmid by: Restriction endonuclease and ⁇ 3 I (purchased from Proraega, USA), the structural gene Rep and / of the adeno-associated virus V genome in the pBR-V2 plasmid ?/(3 ⁇ 40 gene completely excised, the reaction system is: lug pBR-MV2, 10U Bst98 I, 10U Hpa I, 2.5ul 10X buffer D and 19.5 ⁇ 1 deionized water; reaction conditions are: water bath at 37 ° C 4 Then, the nucleotide sequence (CGAATTCATGCGATATCGTT) containing the restriction enzymes EcoR I and EcoR V restriction sites was inserted into the plasmid, and the reaction system was: 500 ng plasmid, 300 ng of the nucleotide sequences of EcoR I and EcoRV, 10 IU of T 4 DNA ligase (purchased from Promega, USA), 1.5 ⁇ l of 10 X
- the TR sequence or the 75th nucleotide sequence of the TR at both ends of the V genome is inserted into a fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability of the r V virus and improve the replication efficiency of the virus.
- reaction system is: ly g plasmid prepared above, 10U Ban I, 1.5 ⁇ 1 10X buffer G and 11.5 ⁇ 1 deionized water; reaction conditions are: water bath at 37 ⁇ 4 hours, then insert 9 nucleotide fragments into the plasmid, the reaction system is: 500ng plasmid, 300ng 9 nucleotide sequence, 10IU T 4 DNA ligase
- reaction conditions were: water bath at 4 ° C for 8 hours.
- Amplification of the CMV promoter and the SV40 promoter by gene amplification by: using the pCI-ne plasmid (purchased from Promega, USA) as a template, in primer 1: AGATCTTCAATATTGGCCAT and Primer 2: The CMV promoter was amplified by PCR under the guidance of TGTCAGAAGCACTGACTGC.
- the PCR amplification conditions were: first 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute, a total of 30 cycles.
- the PCR product was detected by 1.2% agarose gel electrophoresis, and an expected specific band appeared at 740 bp.
- the target band was recovered and purified to obtain CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ° C 40 seconds, a total of 30 cycles; the last 72 ⁇ 8 minutes, after the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, a 359bp appeared The expected specific band was recovered and purified to obtain the SV40 early promoter.
- the beta actin promoter is obtained afterwards.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the full-length Her-2/neu cDNA was amplified by PCR under the guidance of primer 7: ATGGAGCTGGCGGCCTTGTGC and primer 8: TCACACTGGCACGTCCAGACC.
- the PCR amplification conditions were: 94 °C. 4 ⁇ After the end of the reaction, the PCR product was subjected to a 1.2% agarose gel, the reaction product was finished at the end of the reaction.
- Her-2/neu cDNA fragment A was obtained by the same method as above (primer 9) : GTCTTGATCCAGCGGAAC and primer 10: GGACGTCAGAGGGCTGGC), Her-2/neu cDNA fragment B (primer 11: CTGGAAGAGATCAC and primer 12: CCACACATCACTCTG) and Her-2/neu cDNA fragment C (primer 13: CTGGTGACACAGCTTATG and primer 8).
- step A Insert the amplified CMV promoter, SV40 early promoter, beta actin promoter, full-length Her-2/neu cDNA or partial Her-2/neu cDNA fragment into step A by DNA ligation technique.
- the first digestion reaction is carried out, and then the ligation reaction is carried out, wherein the digestion reaction system is: 1 ⁇ g plasmid; 10 U restriction endonuclease rfl and purchased from Promega, USA 2, 5 ⁇ 1 10 X buffer C and 19.
- reaction conditions water bath at 37 ° C for 4 hours
- the reaction system is: 500 ng of the plasmid after digestion, 300 ng promoter DNA, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions: water bath at 4 Torr for 8 hours.
- the plasmid carrying the promoter and the full-length Her-2/neu cDNA or a partial Her-2/neu cDNA fragment were digested with restriction enzymes Nhe I and ⁇ 3 ⁇ 4 ⁇ , respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- recombinant adeno-associated virus vectors (designated rAAV/Her-2/neu) carrying the CMV promoter, SV40 early promoter, beta actin promoter and full-length Her-2/neu cDNA were obtained, respectively, and carried CMV promoter, SV40 early promoter, beta actin promoter and A or B or C different Her-2/neu cDNA fragments (mutant) recombinant adeno-associated virus vectors (named rMV/AmHer- 2/neu, respectively) , rMV/BmHer- 2/neu and rMV/CmHer- 2/neu, collectively named rAAV/mHer- 2/neu).
- the ligated DNA-rAAV/Her- 2/neu and rAAV/mHer-2/neu were separately introduced into genetically engineered E. coli iE. coli DH5 a competent cells (Invitrogen, USA) with 100 g/mL of ampicillin
- E. coli iE. coli DH5 a competent cells Invitrogen, USA
- the LB plate of penicillin was subjected to resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain rAAV/Her-2/neu plasmid and rAAV/mHer-2/neu plasmid.
- the AAV2 vector was obtained, and the vector was digested with restriction endonuclease 3 ⁇ 4i l. After the reaction, the digested product was subjected to 1.2% agarose gel electrophoresis, wherein the detection of rMV/AmHer-2/neu plasmid was carried out. The results are shown in the left panel of Figure 2E (1. DNA molecular weight standards. 2. rAAV/AmHer-2/neu ⁇ EcoR I and BamH I). 3. rAAV/AmHer-2/neu ⁇ EcoR I and Hind 111). 4. rAAV/AmHer-2/neu ⁇ Nhe I and Not 1). 5. rAAV/AmHer-2/neu ⁇ Nhe I and Pst I). 6.
- rAAV/AmHer-2/neu ⁇ Nhe I and Hind U ⁇ 992bp specific band was obtained by EcoR I and BamH I digestion, and 432bp specific strip was obtained by EcoR I and Hind III digestion.
- the specific band of 1512 kb was obtained by Nhe I and ⁇ 3 ⁇ 4 ⁇ I digestion.
- the specific band of 1317 bp was obtained by Nhe I and Pst I digestion, and the specificity of 1320 bp was obtained by Nhe I and Hind III digestion. Sex strips, consistent with the expected results.
- the results of the digestion of rAAV/Her-2/neu and other rMV/mHer-2/neu plasmids were also consistent with the expected results.
- the rMV/Her-2/neu plasmid and the rMV/mHer-2/neu plasmid were further tested by gene amplification (PCR), and the results of the rAAV/raHer-2/neu plasmid were shown in Figure 2E.
- PCR gene amplification
- rAAV/AmHer-2/neu was amplified to obtain the expected specific band of 1503 bp
- rMV/BmHer-2/neu was amplified to obtain the expected specific band of 1512 bp
- rAAV/CmHer-2 /neu was amplified to obtain a desired specific band of 1383 bp.
- Example 5-2 Preparation of recombinant adeno-associated virus (rAAV) and determination of virus titer
- Example 5-1 Construction of a recombinant adeno-associated virus vector carrying the Her-2/neu antigen gene rAAV/Her-2/neu and a recombinant adeno-associated virus vector carrying the Her-2/neu antigen mutant gene rAAV/ mHer-2/neu (rAAV/AmHer- 2/neu, rAAV/BmHer-2/neu and rAAV/CraHer- 2/neu).
- Liposomal transfection reagent Lipofectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus (r V) was prepared by the following method to prepare a virus of a 10. Ocm cell culture dish, and the AAV-HEK293 cells were grown in a carbon dioxide cell incubator to a petri dish. When the area is 70%, proceed as follows:
- Lipofectin 1. 0 ⁇ g rAAV vector (rMV/Her- 2/neu or rAAV/raHer-2/neu), 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin Mix well with 50. 0 ⁇ l of DMEM medium containing 10% fetal bovine serum (or calf serum) and let stand for 20 minutes at room temperature.
- rAAV/Her- 2/ The obtained rAAV virus carrying the full-length cDNA of the tumor-associated antigen gene Her-2/neu antigen gene or a partial Her-2/neu cDNA fragment (A, B, C, mutant gene) was named rAAV/Her- 2/, respectively. Neu, rAAV/AmHer- 2/neu, rAAV/BmHer- 2/neu, rAAV/CmHer-2/neu.
- r V viruses obtained in step one using conventional dot blot hybridization.
- the virus titer assay is performed, and the specific method includes the following steps: Only the DNA probe used is a specific probe for the tumor-associated antigen gene Her-2/neu.
- PCR DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is "Her-2/neu cDNA obtained in Example 5-1, Step C. After PCR amplification, PCR amplification products were subjected to 1.2% agarose gel electrophoresis, and PCR amplification products were detected under ultraviolet light. Band, indicating that the probe is marked successfully.
- the detection result of rMV/AmHer-2/neu is shown in Fig. 4E, and the virus titer of r V/AmHer-2/neu is 10" - 10 12 copies / mL, and the viral drop of rAAV/Her-2/neu The degree is 10 9 - 10 1D copy / mL, the viral titer of rAAV / BmHer-2 / neu is 10 11 - 10 12 copies / mL, and the viral titer of rAAV / CmHer-2 / neu is 10" - 10 12 copies /mL.
- Example 5-3 Tumor-associated antigen-introduced monocyte-macrophage-dendritic cell line killing tumor experiment
- rAAV virus rAAV/Her- 2/neu and rAAV/mHer- 2/neu (rMV/AmHer-2/neu, rAAV/BmHer-2/neu, rAAV/CmHer-2/neu).
- AB1-V Cell Culture Medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF-a) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF-a tumor necrosis factor
- one or more of the rAAV viruses carrying the tumor-associated antigen gene are infected with tumor cells in a tumor-bearing tumor-killing experiment.
- the entire process includes the following steps:
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- the rAAV virus obtained in the embodiment 5-2 of the invention is added in an amount of about 100-1000 M0I, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- the old medium was removed, and the AIM-V medium containing GM-CSF, IL-4 (800 IU/mL) and TNF-a (20 IU/mL) was added, and the culture was continued.
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- the monocyte or immature DC infected with the rAAV of the present invention obtained by the step 1 is labeled with a specific fluorescent antibody (purchased from BD) against the tumor-associated antigen - Her-2/neu using a conventional fluorescent antibody-labeled staining method.
- the number of positive cells was detected by flow cytometry.
- the efficiency of detection of peripheral blood mononuclear cells by recombinant adeno-associated virus rMV/AmHer-2/neu is shown in Fig. 6E, and the efficiency of rMV/AmHer-2/neu infection of peripheral blood mononuclear cells is 95%.
- rAAV/Her-2/neu rAAV/BmHer-2/neu
- rAAV/CmHer-2/neu rAAV/CmHer-2/neu
- DC dendritic cells
- DC expression levels of CD80, CD83, and CD86 are positively correlated with DC function.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- Her- 2/neu protein stimulated DC and non-stimulated DC were controls.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN-y induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured. The cells were harvested, and fluorescent staining was performed by conventional intracellular staining.
- the antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD), and finally the results were measured by flow cytometry.
- the IFN- ⁇ expression level of CTL induced by DC infected with rAAV/CmHer-2/neu is shown in Fig. 8E, and rMV/Her-2/neu and its rAAV carrying the mutant gene (rAAV/AmHer-) 2/neu, rAAV/BmHer-2/neu, rAAV/CraHer-2/neu) DC-induced CTL expression of IFN- ⁇ was significantly higher than that of the control, demonstrating the tumor-associated antigen constructed and prepared by the present invention.
- -Her- 2/neu antigen gene and its mutant gene are highly potent in CTL induced by rAAV-infected DC.
- CTL Cytotoxicity T lymphocyte
- Cytotoxic T lymphocytes are 20: 1 (lymphocytes: tumor cells) and Her-2/neu-positive breast cancers (for example, breast cancer cells, Her-2, colon cancer, lung adenocarcinoma or ovarian cancer) /neu positive cancer cells were mixed, and the activity of CTL killing tumor cells was examined by the conventional MTT method and the 51 Cr (chromium-51) killing test.
- the tumor cell killing rate statistics of CTLs induced by DCs infected with rAAV/AmHer-2/neu are shown in Fig. 9E, and the tumors constructed and prepared by the present invention are compared with CTLs induced by non-stimulated DCs.
- Related antigen-Her- 2/neu antigen and its mutation The CTL induced by the rAAV-infected DC of the type gene can lyse (kill) the tumor cells more effectively, and the killing rate is 50% or more.
- r V-DC the CTL induced by the rAAV-infected DC (collectively referred to as r V-DC) carrying the tumor-associated antigen-Her-2/neu antigen and its mutant gene of the present invention is for breast cancer, colon cancer, lung gland Her-2/neu-positive malignant tumors such as cancer and ovarian cancer have good curative effect and can be used for preparation of anti-tumor drugs.
- the recombinant adeno-associated virus-dendritic cell technique is applied to the rAAV of the present invention in Example 5-3 (r AAV/Her- 2/neu, r V/AmHer-2/neu, rAAV/BmHer-2/neu, Infusion of CTL induced by one or two infected DCs (rAAV-DC) in rAAV/CmHer- 2/neu) 19 cases of breast cancer, colon cancer, lung adenocarcinoma and ovarian cancer patients, infusion volume of CTL For 1 X 10 9 -5 X 10 9 , treatment course: usually 6 months, 2_3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to once every 1-3 months.
- Therapeutic effect (reaction after rAAV-DC treatment)
- B serum tumor markers decreased or disappeared.
- Q The patient's quality of life improved. If the pain is reduced or disappeared, the appetite is increased, etc.
- C CT or PET-CT shows that the cancer lesions or metastatic lesions are significantly reduced or disappeared.
- Adverse reactions In most cases, mild flu-like reactions occur within a short period of time after treatment, but the patients can withstand, and the symptoms disappear in a short period of time. No serious adverse reactions and toxic reactions were observed.
- the course of treatment and survival time are shown in Table 5-2 (survival time after treatment: survival time after the patient started receiving rAAV-DC treatment (when the death case was counted to death).
- rAAV-DC CTL induced by the rAAV-infected DC of the present invention
- Clinical rAAV carries Her- 2/neu rAAV-DC treatment, after treatment, current diagnosis, treatment effect
- the results of imaging observations of changes in metastatic lesions before and after CTL treatment induced by -2/neu-infected DC are shown in Figure 10E (left panel: before treatment, right panel: after treatment for 4 months).
- DCs infected with two or more of rAAV (rAAV/Her- 2/neu, AAV/AmHer-2/neu, rAAV/BraHer-2/neu, rAAV/CraHer-2/neu) of the present invention ( rAAV-DC)
- rAAV-DC DCs infected with two or more of rAAV (rAAV/Her- 2/neu, AAV/AmHer-2/neu, rAAV/BraHer-2/neu, rAAV/CraHer-2/neu) of the present invention
- results DCs infected with two or more of rMV (rMV/Her-2/neu, rMV/AmHer-2/neu, rMV/BmHer-2/neu, rAAV/CmHer-2/neu) of the present invention (rAAV-DC)
- the serum-associated antigen CA27.29 level in the serum decreased significantly, indicating that the tumor burden in the patient was significantly reduced (the tumor cells were significantly reduced), further demonstrating that the rAAV was infected by the present invention.
- the CTL induced by DC can effectively inhibit the growth of malignant tumor cells or kill tumor cells in patients, and can be used for preparing antitumor drugs.
- Part VI Recombinant adeno-associated virus vector r V/ LMP-1
- Example 6-1 Construction and detection of recombinant adeno-associated virus vector r V/LMP-1 and rAAV/mLMP-1
- A. pBR322 plasmid carrying V2 type whole genome DNA (designated pBR-AAV2): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector : transduction of neomycin resistance into mammalian ti ssue culture cel ls. Proc. Natl. Acad. Sci. USA 81 : 6466-6470.
- Human nasopharyngeal carcinoma cells Isolated from cancer tissues of patients with nasopharyngeal carcinoma or purchased from commercial sources, immunohistochemistry confirmed LMP-1 positive.
- the pCI-ne plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid carrying the SV40 early promoter, PSG424, was purchased from Clonit ic, USA.
- the recombinant adeno-associated virus vector carrying the LMP-1 gene or its mutant gene of the present invention is constructed by the following method (shown in Figure 1), and the specific process comprises the following steps:
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and Ecom, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions: water bath at 4 ° C for 8 hours.
- the complete TR sequence at both ends is retained or the 75th nucleotide sequence of the TR at both ends of the AAV genome is inserted into a fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability of the r V virus and
- CTGCGCTGG 9 nucleotides
- the method is as follows: First, the restriction endonuclease I (purchased from Promega, USA) was used to cleave the TR at both ends, and the reaction system was: lwg The plasmid prepared above, 10 U Ban I, 1. 5 ⁇ 1 10 X Buffer G and 11.
- reaction conditions water bath at 37 ° C for 4 hours, then insert 9 nucleotide fragments into the plasmid, the reaction system is: 500ng plasmid, 300ng 9 nucleosides Acid sequence, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions: water bath at 4 ° C 8 hour.
- Amplification of the CMV promoter, SV40 early promoter by gene amplification technology (polymerase chain reaction, PCR).
- the specific method is as follows: Firstly, the CMV promoter is PCR-amplified under the guidance of primer 1 : AGATCTTCAATATTGGCCAT and primer 2 : TGTCAGAAGCACTGACTGC using pCI-neo plasmid (purchased from Promega, USA).
- the PCR amplification conditions are as follows: 94 ° C 4 minutes; further 94 ° C 30 seconds, 60 ° C 35 seconds, 72 ° C 1 minute, a total of 30 cycles; finally 72 ° C 8 minutes, after the reaction, the PCR product was 1.2% agarose gel After electrophoresis detection, an expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ° C 40 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, An expected specific band appeared at 359 bp, and the target band was recovered and purified to obtain the SV40 early promoter.
- the PCR amplification conditions were: 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 58 °C 35 seconds, 72 °C 1 minute 20 seconds, a total of 30 cycles; After the reaction was completed at 72 ° C for 8 minutes, the PCR product was subjected to 1.2% agarose gel electrophoresis, and an expected specific band appeared at 1176 bp. The target band was recovered and purified to obtain beta muscle. The kinesin promoter. The isolated mRNA was reverse transcribed into its cDNA and used as a template. The full-length PSA cDNA was amplified by PCR under the guidance of primer 7: ATTCCGCCGGAGAGCTGTG and primer 8: CCCAGGACACAGAGAGAGGAC.
- the PCR amplification conditions were as follows: 94 ° C for 4 minutes. ; 94 ° C 30 seconds, 59 ° C 35 seconds, 72 ° C 1 minute 15 seconds, a total of 30 cycles; the last 72 ⁇ 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection An expected specific band appeared at 1006 bp, and the band of interest was recovered and purified to obtain a beta actin promoter.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the full-length LMP-1 cDNA was amplified by PCR under the guidance of primer 7: ATGGAACGCGACCTTGAGAG and primer 8: TTAGTCATAGTAGCTTAG.
- the PCR amplification conditions were: 94 ° C for 4 minutes. ; 94 ° C 1 minute, 60 ° C 1 minute, 72 ° C 1 minute 30 seconds, a total of 30 cycles; the last 72 ⁇ 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection An expected specific band appeared at 1370 bp, and the desired band was recovered and purified to obtain a full-length LMP-1 cDNA.
- LMP-1 cDNA fragment A was obtained by the same method as above (primer 7 and primer 9: TCATCAGTAGGAGTAGACC) , LMP-1 cDNA fragment B (primer 10: TCACCCTCCTACTTCATCG and primer 11: CAAGTAAGCAGCCAAAGATG), LMP-1 cDNA fragment C (primer 13: TCTTAGGTCTCTGGATCTAC and primer 8).
- reaction conditions water bath at 37 Torr for 4 hours
- the reaction system is: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU of T 4 DNA Ligase (purchased from Proraega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions: water bath at 4 ° C for 8 hours.
- the plasmid carrying the promoter and the full-length LMP-1 cDNA were digested with restriction enzymes Xba I and BamH I, respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length LMP-1 cDNA (designated rAAV/LMP-1), and the CMV promoter were carried, respectively.
- SV40 early promoter, beta actin promoter and recombinant adeno-associated virus vector of A or B or C different LMP-1 cDNA fragments (mutant) named rAAV/AraLMP-1, rAAV/BmLMP-1, and rMV/CmLMP-1, uniformly named rAAV/mLMP-1).
- the ligated DNA-rAAV/LMP-1 and rAAV/mLMP-1 were separately introduced into genetically engineered Escherichia coli (E. coli DH5 a competent cells (Invitrogen, USA) with 10 ( ⁇ g/mL ampicillin)
- E. coli DH5 a competent cells Invitrogen, USA
- 10 ⁇ g/mL ampicillin
- the LB plate was subjected to resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain r V/LMP-1 plasmid and rAAV/mLMP-1 plasmid.
- the purified rMV/LMP-1 plasmid and r V/mLMP-1 plasmid obtained in the first step were subjected to restriction enzymes (in order, Xba I BamH I, Xba I Not I, EcoR Sal I, EcoR BamH I, Pst I) was digested and the restriction enzymes used were purchased from Promega, USA.
- the r V vector without LMP-1 gene was used as a negative control (the CMV promoter, the SV40 early promoter, and the beta actin promoter were sequentially inserted into the modified pBR-V2 vector of step A, and the vector was used for restriction.
- rAAV/LMP-1 ( ⁇ 3 ⁇ 4 ⁇ ⁇ endonuclease). 6. DNA molecular weight standards. ), Xba I. and BamH I were digested to obtain a specific band of 1370 bp, and a 1370 kb specific band was obtained by digestion with Jba I and ⁇ 3 ⁇ 4 ⁇ I. The specificity of 2213 bp was obtained by EcoR V and S&1 I digestion. The 876 bp specific band was obtained by digestion with EcoR V and BamH I. The specific bands of 684 bp and 1003 bp were obtained by Pst I digestion, which was consistent with the expected results.
- the results of the digestion assay of the rAAV/mLMP-1 plasmid were also consistent with the expected results.
- the rAAV/LMP-1 plasmid and the rAAV/mLMP-1 plasmid were further tested by gene amplification (PCR), and the results of the rAAV/LMP-1 plasmid were shown in the right panel of Figure 2F (1.
- PCR amplification product of LMP-1 cDNA 3. Positive control.) 1370 bp of the expected specific band was obtained by amplification.
- PCR results of the rAAV/mLMP-1 plasmid were also in agreement with the expected results (the size of the amplified product was rAAV/AmLMP-1: 268 bp, rAAV/BmLMP-1: 87 bp, rAAV/CmLMP-1: 860 bp).
- the above detection results indicated that the recombinant adeno-associated virus vector rAAV/LMP-1 carrying the LMP-1 gene and the recombinant adeno-associated virus vector rAAV/mLMP-l carrying the LMP-1 mutant gene were obtained with the correct insertion position and sequence.
- Example 6-2 Preparation of recombinant adeno-associated virus (rMV) and determination of virus titer
- the recombinant adeno-associated virus vector rAAV/LMP-1 carrying the LMP-1 gene and the recombinant adeno-associated virus vector carrying the LMP-1 mutant gene rAAV/mLMP-1 ( rAAV/AraLMP - 1. rAAV/BmLMP-1, rAAV/CmLMP-1 and rAAV/DmLMP-1).
- V-HEK293 cells containing adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the chromosome of the cell: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva) -Internati, M., Grizzi, F. Salati, E., Roman, JJ, Lim S., and Hermonat, PL Relax induction of cytotoxic T cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic Cells by an adeno-as soc i at ed virus vector. Cancer Gene Therapy 8 : 948-957. ).
- adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the chromosome of the cell: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva) -Internati, M., Grizzi,
- Liposomal transfection reagent Lipofectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- recombinant adeno-associated virus was prepared by the following method to prepare a plate of 10. 0 cm cell culture.
- the virus of the dish was taken as an example, when AAV-HEK293 cells were grown in a carbon dioxide cell incubator to approximately culture.
- the dish area is 70%, proceed as follows:
- Lipofectin 1.0 ⁇ g rAAV vector (rMV/LMP-1 or rAAV/mLMP-1), 1. 0 ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50 ⁇ 0 ⁇ 1 DMEM medium containing 10% fetal bovine serum (or calf serum) was mixed and allowed to stand at room temperature for 20 minutes.
- r AAV/LMP-1 The obtained rAAV viruses carrying the full-length LMP-1 cDNA of the tumor-associated antigen gene LMP-1 gene or a partial LMP-1 cDNA fragment (A, B, C mutant gene) were named r AAV/LMP-1, rAAV/, respectively. AmLMP-1, rAAV/BmLMP-1, r AAV/CmLMP-1.
- the various r V viruses (r V/LMP-l, rAAV/AmLMP-1, rAAV/BmLMP-1, rAAV/CmLMP-l) obtained in step 1 were tested for virus titer.
- the method comprises the following steps: Only the DNA probe used is a specific probe for a tumor-associated antigen gene. .
- the rMV virion DNA was extracted using a conventional DNA phenol/chloroform extraction method.
- B The nylon membrane was placed in a dot blotter, and the alkali-denatured r V virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- D Prepare a DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is "LMP-1 cDNA obtained in Step 6-1 of Example 6-1.
- PCR After PCR amplification, PCR The amplified product was subjected to 1.2% agarose gel electrophoresis, and the PCR amplification product was detected under ultraviolet light, and a positive band appeared, indicating that the probe was successfully labeled.
- r V/LMP-1 the detection result of r V/LMP-1 is shown in Fig. 4F
- the virus titer of rAAV/LMP-1 is ⁇ - ⁇ " 1 copy/mL
- the virus titers of the three rMV/AmLMP-1 are 10 11 - 10'° copy/mL.
- Example 6-3 Tumor-associated antigen-introduced monocyte-macrophage-dendritic cell line killing tumor experiment
- rAAV virus rMV/LMP-1 and rAAV/mLMP-1 (rAAV/AmLMP-1, rAAV/BmLMP-U rAAV/CmLMP-1).
- AB1-V Cell Culture Medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the whole process of killing tumors based on the monocyte-based tumor of a tumor patient infected with one or more rAAV viruses carrying a tumor-associated antigen gene includes The following steps:
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- Example 6-2 of the present invention Adding one (or more, better effect)
- the rAAV virus obtained in Example 6-2 of the present invention is added in an amount of about 100-1000 MOI, and then GM-CSF (800 IU/mL) is added, and the culture is continued. hour.
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- a specific fluorescent antibody (purchased from BD, USA) against the tumor-associated antigen LMP-1 was used to label the mononuclear cells or immature DCs infected with the r V of the present invention obtained in the first step, The number of positive cells was detected by flow cytometry. Among them, the efficiency of detection of peripheral blood mononuclear cells by recombinant adeno-associated virus r V/LMP-1 is shown in Fig. 6F, and the efficiency of rAAV/LMP-1 infection of peripheral blood mononuclear cells is 87%, constructed and prepared.
- the efficiency of rAAV (VrAAV/AmLMP - 1, VrAAV/BmLMP-1, VrAAV/CmLMP-1) carrying tumor-associated antigens in peripheral blood mononuclear cells is about 90%, that is, about 90% of the periphery. Blood mononuclear cells can be infected with rAAV virus, demonstrating that the rAAV of the present invention has a high infection efficiency.
- DC dendritic cells
- DC expression levels of CD80, CD83, and CD86 are positively correlated with DC function.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- LMP- 1 protein stimulated DC and non-stimulated DC were controls.
- the expression of CD80, CD83 and CD86 in DCs infected with recombinant adeno-associated virus rMV/LMP-1 is shown in Figure 7F, and is expressed by VrMV/LMP-1 and other rMVs carrying tumor-associated antigens (rAAV/AmLMP-1).
- rAAV/BmLMP-1, rAAV/CmLMP-1) The level of CD molecules expressed by DCs was significantly higher than that of the control, demonstrating the construction and preparation of rAAV-infected peripheral blood cells carrying the tumor-associated antigen LMP-1 and its mutant gene. After nuclear cells, the induced DCs are powerful.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- y.
- the level of CTL-expressing IFN- ⁇ induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry.
- the IFN- ⁇ expression level of CTL induced by DCs infected with rAAV/LMP-1 is shown in Fig. 8F, and is rAAV/LMP-1 and other rAAVs carrying tumor-associated antigens (rAAV/AmLMP-1, rAAV/).
- BmLMP-K rAAV/CmLMP-1) The level of CTL expression of IFN- y induced by infected DC was significantly higher than that of the control, demonstrating the tumor-bearing resistance constructed and prepared by the present invention.
- the CTLs induced by r-infected DCs of pro-LMP-1 and its mutant genes are powerful.
- CTL Cytotoxicity T lymphocyte
- the cytotoxic T lymphocytes induced by DCs infected with rMV were 20 : 1 (lymphocytes: tumor cells)
- the activity of CTLs to kill tumor cells was examined by conventional MTT assay and 51 Cr (chromium-51) killing assay.
- the statistical results of the tumor cell killing rate of CTLs induced by rAAV/LMP-1 infected DCs are shown in Fig.
- rMV antigen-negative breast cancer, colon cancer, lung adenocarcinoma, and prostate cancer cells
- rMV rAAV/LMP-1, rAAV/AmLMP-1, rAAV/BmLMP-1).
- rAAV/CmLMP-U The specificity of cytotoxic T lymphocytes induced by infected DCs to kill tumor cells. Among them, the specific detection results of tumor cell killing of CTL induced by rAAV/LMP-1 infected DC are shown in FIG. 9F, and have no killing effect on LMP-1 antigen-negative cancer cells, demonstrating the carrying and constructing of tumors constructed and prepared by the present invention.
- the CTL induced by the rAAV-infected DC of the related antigen-LMP-1 and its mutant gene has antigen specificity, that is, no killing effect on antigen-negative cells.
- rAAV-DC rAAV-infected DC
- rAAV-DC rAAV-infected DC
- LMP-1 antigen-positive nasopharyngeal carcinoma LMP-1 antigen-positive nasopharyngeal carcinoma. It has good curative effect and can be used to prepare anti-tumor drugs.
- rAAV/LMP-U rAAV/AmLMP-K rAAV/BmLMP-1 and rAAV/CmLMP-1) in Example 6-3
- CTLs induced by two infected DCs were returned to 8 patients with nasopharyngeal carcinoma, and the infusion volume was IX 10 9 -5 X 10 9 .
- Treatment course usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to once every 1-3 months.
- r V-DC Recombinant adeno-associated virus-dendritic cell technology
- the imaging changes of 8 cases of nasopharyngeal carcinoma patients in the first step before and after treatment were observed.
- One of the patients with stage IV metastatic nasopharyngeal carcinoma was induced by rAAV/LMP-1 infected DCs.
- the results of the imaging observations of the case are shown in Fig. 10F, and the results were infected with one or both of the rAAV (rAAV/LMP-K rAAV/AmLMP-1, rAAV/BmLMP-1 and rAAV/QnLMP-1) of the present invention.
- DC rAAV-DC After the CTL treatment induced, the metastatic lesion of the patient disappeared significantly, further demonstrating that the CTL induced by the DCA-infected DC of the present invention can effectively inhibit the growth of malignant cells or kill the tumor in the patient.
- Cells which can be used to prepare anti-tumor drugs.
- Example 7-1 Construction and detection of recombinant adeno-associated virus vector rAAV/BA46 and rAAV/mBA46 Materials and sources:
- A. pBR322 plasmid carrying AAV type 2 whole genome DNA (designated pBR-V2): prepared by Professor Paul L. Hermonat, one of the main technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81: 6466-6470.
- B. Human breast cancer cells isolated from cancerous tissues of breast cancer patients, immunohistochemically confirmed BA46 positive (or purchased from commercial sources).
- the pCI-neo plasmid carrying the CMV promoter was purchased from Promega, USA, and the plasmid pSG424 carrying the SV40 early promoter was purchased from Clonitic, USA.
- the recombinant adeno-associated virus vector carrying the BA46 gene or its mutant gene of the present invention is constructed by the following method (as shown in Fig. 1), and the specific process comprises the following steps:
- reaction system is: lg pBR- AAV2, 10U Bst98 I, 10U Hpa I, 2.5 ⁇ 1 10X. Buffer D and 19.5 ⁇ 1 deionized water; reaction conditions are: at 37° C water bath for 4 hours.
- the nucleotide sequence (CGAATTCATGCGATATCGTT) containing the restriction enzymes EcoR 1 and EcoR cleavage sites was inserted into the plasmid.
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and EcoR N, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1.5 ⁇ l 10 XT 4 DNA ligation buffer and 11.5 ⁇ l of deionized water; reaction conditions were: water bath at 4 ° C for 8 hours. Then, retain the complete TR sequence at both ends or insert the 75th nucleotide sequence of the TR at both ends of the AAV genome.
- CTGCGCTGG Fragment consisting of 9 nucleotides: CTGCGCTGG, the purpose of which is to improve the stability of rAAV virus and improve the replication efficiency of the virus by first cutting the TR at both ends with restriction endonuclease I (purchased from Proraega, USA)
- the reaction system is: lwg the above prepared plasmid, 10U Ban I, 1. 5 l 10 X buffer G and 11. 5 ⁇ 1 deionized water; the reaction conditions are: water bath at 37 4 for 4 hours, then 9 cores The cleavage fragment was inserted into the plasmid.
- the reaction system was: 500 ng plasmid, 300 ng 9 nucleotide sequence, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11 5 ⁇ l of deionized water; reaction conditions: water bath at 4 ° C for 8 hours.
- PCR polymerase chain reaction
- primer 1 AGATCTTCAATATTGGCCAT
- primer 2 TGTCAGAAGCACTGACTGC with pCI- neo plasmid (purchased from Promega, USA)
- the PCR amplification conditions are: 94 ° C first 4 minutes; further 94 ° C 30 seconds, 60 ° C 35 seconds, 72 ° C 1 minute, a total of 30 cycles; finally 72 ° C 8 minutes, after the reaction, the PCR product was 1.2% agarose gel After electrophoresis detection, an expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ⁇ 40, seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, at 359 bp An expected specific band appeared, and the target band was recovered and purified to obtain the SV40 early promoter.
- the recombinant adeno-associated virus vector of the present invention is specifically prepared by separating nucleic acid and mRNA from human breast cancer cells by using nucleic acid separation technology (the total DNA and mRNA can also be obtained synthetically or commercially), and then the total DNA is The template, PCR-amplified beta actin promoter under the guidance of primer 5: CCCGGGCCCAGCACCCCAAG and primer 6: CATCCATGGTGAGCTGCG, the PCR amplification conditions were: 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 58 ° C for 35 seconds 72 ° C 1 minute 20 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, an expected specific strip appeared at 1176 bp With the strip, the target strip is recovered and purified.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the full-length BA46 cDNA was amplified by PCR under the guidance of primer 7: CCGCAGCATGCCGCGCCC and primer 8: CACTAACAGCCCAGCAGC.
- the PCR amplification conditions were: First, 94 ° C for 4 minutes; then 94 ° C for 30 seconds, 60 ° C for 35 seconds, 72 ° C for 1 minute and 30 seconds, a total of 30 cycles; the last 72 ° C for 8 minutes, after the reaction, the PCR product was 1.
- a 2% agarose gel electrophoresis assay showed an expected specific band at 1173 bp.
- BA46 cDNA fragment A was obtained in the same manner as above (primer 7 and primers).
- 9 TGCGCGGTTCAGGCGGTTC
- BA46 cDNA fragment B (primer 10: GGCATGGTCAATGCCTGGAC and primer 11: CTTCAGGCCCAGGGGATT)
- BA46 cDNA fragment C (primer 12: AATAACAGCATCCCTGACAAGC and primer 8).
- the above amplified CMV promoter, SV40 early promoter, beta actin promoter, full-length BA46 cDNA or partial BA46 cDNA fragment are sequentially inserted into the modified pBR-AVV2 vector of step A,
- the first digestion reaction is carried out, and then the ligation reaction is carried out, wherein the digestion reaction system is: lg plasmid; 10U restriction endonuclease ⁇ 3 ⁇ 43 ⁇ 471 and 53 ⁇ 47 I (purchased from Promega, USA), 2.5 ⁇ 1 10X Buffer C and 19.5 ⁇ l of deionized water; reaction conditions were: water bath at 37 ° C for 4 hours, the reaction system was: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU of T 4 DNA ligase (purchased from Promega, USA, 1.5 ⁇ 1 10XT 4 DNA ligation buffer and 11.5 ⁇ l deionized water; reaction conditions
- the plasmid carrying the promoter and the full-length BA46 cDNA were digested with restriction enzymes ⁇ 3 ⁇ 4 I and ⁇ 3 ⁇ 4 ⁇ I, respectively.
- restriction enzymes ⁇ 3 ⁇ 4 I and ⁇ 3 ⁇ 4 ⁇ I respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length BA46 cDNA designated rMV/BA46
- the CMV promoter, SV40 promoter, and beta were obtained.
- Actin promoter and A or B or C different BA46 cDNA fragments (mutant) recombinant adeno-associated virus vectors (named r V/AmBA46, r V/BmBA46 and r V/CmBA46, respectively, collectively named rMV/mBA46 ).
- the ligated DNA-rAAV/BA46 and r V/mBA46 were introduced into genetically engineered E. coli (K coli) DH5a competent cells (Invitrogen, USA), respectively, using LB plates containing 10 ( ⁇ g/mL ampicillin). For resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain rAAV/BA46 plasmid and rAAV/mBA46 plasmid.
- the purified rAAV/BA46 plasmid and the rAAV/mBA46 plasmid obtained in the first step were digested with restriction enzymes (in turn, ⁇ s I, Nhe ⁇ Not I, Nhe I Pst I, Nhe J coR), and the restriction was used.
- restriction enzymes in turn, ⁇ s I, Nhe ⁇ Not I, Nhe I Pst I, Nhe J coR
- the endonuclease was purchased from Promega, USA.
- the digested product was detected by 1.2% agarose gel electrophoresis, and the detection result of rAAV/BA46 plasmid is shown in the left figure of Figure 2G (1. DNA molecular weight standard 2.
- rAAV/BA46 (Nhe l ⁇ iNot l endonuclease) 4.
- rAAV/BA46 (Nhe I and Pst I endonuclease) 5.
- rAAV/ BA46 (Nhe I and EcoR V endonuclease).
- the specific bands of 361 bp and 789 bp were obtained by digestion with cadaveric si I.
- the specific band of 1179 bp was obtained by Nhe I and Not I digestion, and Nhe I was obtained.
- a 1457 bp specific band was obtained by digestion with Pst I, and a specific band of 1790 bp was obtained by Nhe I and EcoR digestion, which was consistent with the expected results.
- the PCR detection of rAAV / raBA46 plasmid The results are also consistent with the expected results (the size of the amplified products is in order rAAV/AmBA46: 340 bp, rAAV/BmBA46: 378 bp rAAV/CmBA46: 453 bp).
- Example 7-2 Preparation of recombinant adeno-associated virus (r V) and determination of virus titer
- Liposomal transfection reagent Lipofectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 1.0 cm cell culture dish, and when MV-HEK293 cells were grown in a carbon dioxide cell incubator to about a culture dish area At 70%, do the following:
- A. follow the instructions for Lipofectin: l. O yg rAAV vector (rAAV/BA46 or rAAV/mBA46), l. O ug pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50. 0 ⁇ 1 with 5 % fetal
- the bovine serum (or calf serum) was mixed in DMEM medium and allowed to stand at room temperature for 20 minutes.
- the virus titer was determined by routine dot blot hybridization of various rAAV viruses (r V/BA46, rAAV/AmBA46, rAAV/BmBA46, rAAV/CmBA46) obtained in step 1.
- the specific method includes the following steps:
- the probe is a specific probe for the tumor antigen gene.
- the rAAV virus particle DNA was extracted using a conventional DNA phenol/chloroform extraction method.
- the nylon membrane was placed in a dot blotter, and the alkali-denatured rAAV virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- Example 7-1 Step C.
- PCR DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is the BA46 cDNA obtained in Example 7-1, Step C.
- PCR amplification After PCR amplification, PCR amplification The product was subjected to 1.2% agarose gel electrophoresis, and the PCR amplification product was detected under ultraviolet light, and a positive band appeared, indicating that the probe was successfully labeled.
- rAAV/BA46 the detection results of rAAV/BA46 are shown in Fig. 4G, the virus titer of rAAV/BA46 is ⁇ 12 copies/mL, and the virus titers of the three rAAV/mBA46 are all 10 11 - 10 12 copies/mL.
- Example 7-3 Tumor antigen-introduced monocyte-macrophage-dendritic cell line killing tumor experimental materials and sources thereof:
- rAAV virus rAAV/BA46 and rMV/mBA46 (rAAV/AmBA46, rAAV/BmBA46, rAAV/CmBA46).
- AIM-V cell culture medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the whole process of killing a tumor-based tumor-infecting tumor in a patient with one or more viruses carrying a tumor antigen gene includes the following steps:
- peripheral blood cell separator or lymphocyte separation solution
- AIM-V medium PBMC
- cell culture flask Incubate for 2 hours in a constant temperature carbon dioxide incubator.
- Example 7-2 of the present invention remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, ie, peripheral blood lymphocytes, were mixed with AIM-V medium and cultured for further use.
- C. Adding one (or more, better effect) The rMV virus obtained in Example 7-2 of the present invention is added in an amount of about 100-1000 MOI, and then GM-CSF (800 IU/mL) is added, and the culture is continued for 4 hours. .
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- the specific fluorescent antibody against the tumor antigen-BA46 (purchased from BD, USA) was used to label the mononuclear cells or immature DCs infected with the r V of the present invention obtained in the first step.
- Flow cytometry was used to detect the number of positive cells.
- the efficiency of detection of peripheral blood mononuclear cells by recombinant adeno-associated virus rMV/BA46 is shown in Fig. 6G, and the efficiency of rAAV/BA46 infection of peripheral blood mononuclear cells is about 91%, and various tumor antigens are constructed and prepared.
- rMV rAAV/AmBA46, rAAV/BmBA46, rAAV/CmBA46
- DC dendritic cells
- DC expression levels of CD80, CD83, and CD86 are positively correlated with DC function.
- the detection method was to detect the levels of DC expression CD80, CD83 and CD86 obtained in step one using fluorescently labeled antibodies against these three CD molecules (purchased from BD, USA). BA46 protein stimulated DC and non-stimulated DC were controls.
- CD80, CD83 and CD86 in DCs infected with recombinant adeno-associated virus rAAV/BA46 is shown in Figure 7G, and rAAV/BA46 and other rAAVs carrying tumor antigens (rMV/AmBA46, rAAV/BmBA46, rAAV/) CmBA46)
- rMV/AmBA46, rAAV/BmBA46, rAAV/) CmBA46 The level of CD molecules expressed by infected DCs was significantly higher than that of the control, demonstrating that the DCs induced by the constructed and prepared rAAV carrying the tumor antigen-BA46 and its mutant gene infecting peripheral blood mononuclear cells are powerful.
- C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells are positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN- ⁇ induced by DCs infected with the rMV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescently labeled antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry.
- the expression level of IFN- ⁇ of CTL induced by DC infected with rAAV/BA46 is shown in Fig. 8G, and it is rMV/BA46 and other rAAV carrying tumor antigen (rAAV/AmBA46, rAAV/BmBA46, r AAV/CmBA46).
- the level of CTL expression of IFN- ⁇ induced by infected DC was significantly higher than that of the control, demonstrating that CTLs induced by the rAAV-infected DC carrying the tumor antigen- ⁇ 46 gene and its mutant gene constructed and prepared by the present invention are powerful.
- CTL Cytotoxic T lymphocyte
- the cytotoxic T lymphocytes induced by DCs infected with rAAV were 20:1 (lymphocytes: tumor cells).
- the activity of CTL killing tumor cells was examined using a conventional sputum method and a sl Cr (chromium-51) killing test. The statistical results of tumor cell killing rate of CTL induced by DC infected with rAAV/BA46 are shown in Fig.
- the BA46-negative cervical cancer, colon cancer, and lung adenocarcinoma cells were used as controls.
- the same method was used to detect DCs infected with rAAV (rAAV/BA46, rAAV/AmBA46> rMV/BmBA46, r AAV/CmBA46) in the first step.
- the specificity of the induced cytotoxic T lymphocytes killing tumor cells is shown by FIG. 9G, and is induced by the DCA-infected DC carrying the tumor antigen-BA46 and its mutant gene constructed and prepared by the present invention.
- the CTL has no killing effect on BA46-negative cancer cells, and it is proved that the CTL induced by the rAAV-infected DC carrying the tumor antigen-BA46 gene and its mutant gene constructed and prepared by the present invention has antigen specificity, that is, antigen-negative The cells have no killing effect.
- r V-DC rAAV-sensing DC
- Recombinant adeno-associated virus-dendritic cell technology which is the DC of Example 7-3 infected with one or both of the rAAVs of the invention (rAAV/BA46, rAAV/AmBA46, rAAV/BmBA46 and rAAV/CmBA46) (rAAV-DC) induced CTL reinfusion in 10 patients with breast cancer, the infusion volume was 1 X 10 9 - 5 X 10 9 .
- Treatment course Usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to 1 to 3 months.
- rAAV-DC CTL induced by the rAAV-infected DC of the present invention
- the patient's metastatic lesions apparently disappeared, further demonstrating that the CTL induced by the DCA-infected DC of the present invention can effectively inhibit the growth of malignant cells or kill tumor cells in the patient, and can be used for preparing an antitumor drug.
- Example 7-3 The fluorescent antibody-labeled staining method in Example 7-3 was used to detect the level of serum CA27.29 tumor-associated antigen in the serum of 10 patients with breast cancer before and after treatment in the first step (data from the test results of the experimental hospital), The changes of serum CA27.29 tumor-associated antigen levels in breast cancer patients before and after CTL treatment induced by rAAV/BA46-infected DCs are shown in Fig.
- Example 8-1 Construction and Detection of Recombinant Adeno-associated Virus Vectors rAAV/AFP and rAAV/mAFP Materials and Sources -
- A. pBR322 plasmid carrying AAV type 2 whole genome DNA (designated pBR-AAV2): prepared by Professor Paul L. Hermonat, one of the principal technical and technical leaders of Beverly International Co., Ltd. (Hermonat, PL, and Muzyczka, N. Use of adeno- associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc. Natl. Acad. Sci. USA 81: 6466-6470.
- Human HepG2 cell line It is derived from the American Cell and Tissue Collection Center and expresses alpha-fetoprotein antigen (AFP); it is also commercially available.
- AFP alpha-fetoprotein antigen
- the pCI-neo plasmid carrying the CMV promoter was purchased from Proraega, USA, and the plasmid carrying the SV40 early promoter, PSG424, was purchased from Clonitic, USA.
- Gene amplification nucleotide primers Designed according to the published alpha-fetoprotein antigen (AFP) gene sequence (US NCI gene bank: NM-001134) published in the US Gene Bank.
- AFP alpha-fetoprotein antigen
- the recombinant adeno-associated virus vector carrying the AFP gene or its mutant gene of the present invention is constructed by the following method (as shown in Fig. 1), and the specific process comprises the following steps:
- the nucleotide sequence containing the restriction enzymes EcoR I and EcoR V cleavage sites was inserted into the plasmid.
- the reaction system was: 500 ng plasmid, 300 ng of nucleotide sequence of EcoR I and EcoR V, 10 IU.
- T 4 DNA ligase purchased from Promega, USA
- 1.5 ⁇ l 10 XT 4 DNA ligation buffer and 11.5 ⁇ l of deionized water reaction conditions were: water bath at 4 Torr for 8 hours.
- the replication efficiency of the virus was as follows: First, the restriction endonuclease I (purchased from Promega, USA) was used to cleave the TR at both ends.
- the reaction system was: lg The plasmid prepared above, 10U Ban I, 1. 5 ⁇ 1 10 X buffer Liquid G and 11.
- reaction conditions water bath at 37 ° C for 4 hours, and then insert 9 nucleotide fragments into the plasmid, the reaction system is: 500ng plasmid, 300ng 9 nucleotides Sequence, 10 IU T 4 DNA ligase (purchased from Promega, USA), 1. 5 ⁇ l 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l of deionized water; reaction conditions were: water bath at 4 Torr for 8 hours.
- the PCR amplification conditions are as follows: 94 ° C 4 minutes; further 94 ° C 30 seconds, 60 ° C 35 seconds, 72 ° C 1 minute, a total of 30 cycles; the last 72 ⁇ 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection An expected specific band appeared at 740 bp, and the target band was recovered and purified to obtain a CMV promoter.
- the PSV424 plasmid (purchased from Clonitic, USA) was used as a template to PCR-amplify the SV40 early promoter under the guidance of primer 3: GAACCAGCTGTGGAATGTGTC and primer 4: TCAGGAAGCTTAGATCTAGC.
- the PCR amplification conditions were: 94 ° C for 4 minutes; °C 30 seconds, 60 ° C 35 seconds, 72 ° C 40 seconds, a total of 30 cycles; the last 72 ° C 8 minutes, after the reaction, the PCR product was subjected to 1.2% agarose gel electrophoresis detection, An expected specific band appeared at 359 bp, and the target band was recovered and purified to obtain the SV40 early promoter.
- PCR amplification of beta actin promoter, full-length AFP cDNA and partial AFP cDNA fragments (named A (from base 5-1277 base), B (from 5' end, 653-1277)
- A from base 5-1277 base
- B from 5' end, 653-1277
- the above fragment is exemplified, but not limited to the above fragment, and other AFP cDNA fragments having the same function as the AFP cDNA can be used for construction.
- the recombinant adeno-associated virus vector of the present invention is specifically prepared by separating nucleic acid and mRNA from human HepG2 cells by nucleic acid isolation technology (also obtained synthetically or commercially), and then using total DNA as a template in primer CCCGGGCCCAGCACCCCAAG And primer 6: CATCCATGGTGAGCTGCG guided PCR amplification of beta actin promoter, PCR amplification conditions: first 94 ° C 4 minutes; then 94 ° C 30 seconds, 58 ° C 35 seconds, 72 ° C 1 minute 20 Seconds, a total of 30 cycles; the last 72 °C 8 minutes, after the end of the reaction, the PCR product was detected by 1.2% agarose gel electrophoresis, an expected specific band appeared at 1176 bp, the target band Recovered and purified to obtain beta actin Mover.
- the mRNA was reverse transcribed into its cDNA and used as a template.
- the full-length AFP cDNA was amplified by PCR under the guidance of primer 7: CTTCCACCACTGCCAATAAC and primer 8: TTGTCTTCTCTTCCCCTG.
- the PCR amplification conditions were: First, 94 ° C for 4 minutes; then 94 ° C for 1 minute, 60 ° 1 minute, 72 ° C for 2 minutes, a total of 30 cycles; the last 72 ° C for 10 minutes, after the reaction, the PCR product was 1.2% agarose By gel electrophoresis, an expected specific band appeared at 1890 bp, and the target band was recovered and purified to obtain a full-length AFP cDNA.
- AFP cDNA fragment A was obtained by the same method as above (primer 7 and primer 9: TGCTTGGCTCTCCTGGATGT) , AFP cDNA fragment B (primer 10: CCAAACAAAGGCAGCAAC and primer 9), AFP cDNA fragment C (primer 10 and primer 8).
- step D Insert the amplified CMV promoter, SV40 early promoter, beta actin promoter, full-length AFP cDNA or partial AFP cDNA fragment into the P BR-V2 vector modified in step A by DNA ligation technology.
- the promoter For inserting the promoter, first performing a digestion reaction, and then performing a ligation reaction, wherein the digestion reaction system is: lg plasmid; 10 U restriction enzyme and I (purchased from Promega, USA), 2. 5 ul 10 X Buffer C and 19.
- reaction conditions water bath at 37 Torr for 4 hours
- the reaction system is: 500 ng of the digested plasmid, 300 ng of promoter DNA, 10 IU of T 4 DNA ligase (purchased from Promega, USA, 1. 5 ⁇ 1 10 XT 4 DNA ligation buffer and 11. 5 ⁇ l deionized water; reaction conditions: water bath at 4 ° C for 8 hours.
- the plasmid carrying the promoter and the full-length AFP cDNA were digested with restriction endonuclease V and Not I, respectively.
- the system and conditions for the digestion reaction and the ligation reaction are the same as described above.
- a recombinant adeno-associated virus vector (designated rMV/AFP) carrying the CMV promoter, the SV40 early promoter, the beta actin promoter and the full-length AFP cDNA, and the CMV promoter carrying the SV40 early promoter were obtained.
- a beta actin promoter and a recombinant adeno-associated virus vector of A or B or C different AFP cDNA fragments (named rAAV/AmAFP, rAAV/BmAFP and rAAV/CmAFP, respectively, named rAAV/mAFP).
- the ligated DNA-rAAV/AFP and rAAV/mAFP were separately introduced into genetically engineered Escherichia coli (£ coif) DH5 a competent cells (Invitrogen, USA), using LB plates containing 10 ( ⁇ g/mL ampicillin). For resistance screening, white single colonies were picked, plasmids were extracted and purified to obtain rAAV/AFP plasmid and rAAV/mAFP plasmid.
- the purified rAAV/AFP plasmid and the rAAV/mAFP plasmid obtained in the first step were subjected to restriction enzymes (in order, Not I BamH I, EcoR I Not I, EcoR Mhe I, EcoR WNot I, Pst I).
- the restriction enzymes used were all purchased from Promega, USA.
- the digested product was subjected to 1.2% agarose gel electrophoresis, and the detection result of the rAAV/AFP plasmid was as shown in the left diagram of Fig. 2H' ( 1. rAAV/AFP ( ⁇ 3 ⁇ 4 ⁇ I and BamH I inscribed) Enzyme) 2.
- rAAV/AFP ⁇ EcoR I and Not I endonuclease 3. rAAV/AFP ⁇ EcoR V and Nhe I endonuclease). 4. rAAV/AFP ⁇ EcoR V and Not I endonuclease). 5. rAAV/AFP (Pst I endonuclease). 6. DNA molecular weight standards. ), cut by ?I and I A specific band of 1305 bp was obtained, and a specific band of 1295 bp was obtained by EcoR i Not I digestion. A specific band of 3568 bp was obtained by EcoR V and Nhe I digestion, and digested with EcoR V and Not I.
- the specific band of 1896b was digested with Pst I to obtain specific bands of 251 bp, 382 bp, 509 bp, 983b and 1613 bp, which were consistent with the expected results.
- the results of the digestion of the rAAV/mAFP plasmid were also consistent with the expected results.
- the rAAV/AFP plasmid and the rAAV/mAFP plasmid were further tested by gene amplification (PCR), and the results of the rAAV/AFP plasmid are shown in the right panel of Figure 2H (1. DNA molecular weight standard. 2. AFP cDNA 3. Negative control.), 1890 bp of the expected specific band was obtained by amplification.
- the PCR results of the rAAV/mAFP plasmid were also consistent with the expected results (the size of the amplified product was rAAV/AmAFP: 1266 bp, rAAV/BmAFP: 625 bp, rAAV/CmAFP: 1250 bp).
- the above results indicated that the recombinant adeno-associated virus vector r V/AFP carrying the AFP gene and the recombinant adeno-associated virus vector rAAV/mAFP carrying the AFP mutant gene were obtained with the correct insertion position and sequence.
- Example 8-2 Preparation of recombinant adeno-associated virus (rAAV) and determination of virus titer
- a helper plasmid containing the AAV gene and the i/ ⁇ 3 ⁇ 4D gene pHelper constructed by Professor Liu Yong, Gene Therapy Center, affiliated University of Arkansas Medical School (Liu, Y., Chiriva-Internati, M., Grizzi; F. SER s s s s s s s Cancer Gene Therapy 8 : 948-957. )
- AAV-HEK293 cells containing adenoviral genes (El, E2A, E4, VAI and VAII genes) integrated into the chromosome of the cell: established by the Gene Therapy Center of the University Hospital of Arkansas Medical School (Liu, Y., Chiriva-Internati, M., Grizzi, F. Salat i, E., Roman, JJ, Lim S., and Herraonat, PL Rapid induction of cytotoxic T cell response against cervical cancer cells by human papi l lomavirus type 16 E6 antigen gene Del ivery into human dendritic cel ls by an adeno- associated virus vector. Cancer Gene Therapy 8 : 948 - 957. ).
- Liposomal transfection reagent Lipof ectin purchased from Invotrogen, USA.
- PCR DIG Labeling Kit and DIG Hybridization Assay Kit purchased from Roche, Switzerland.
- a recombinant adeno-associated virus was prepared by the following method to prepare a virus of a 1.0 cm cell culture dish, and the AAV-HEK293 cells were grown in a carbon dioxide cell incubator to an area corresponding to the culture dish. At 70%, do the following:
- Lipofectin l. O yg rAAV vector (rMV/AFP or rAAV/mAFP), ⁇ . ⁇ ⁇ g pHelper plasmid, 4. 0 ⁇ 1 Lipofectin and 50. 0 ⁇ 1 with 5%
- the fetal bovine serum (or calf serum) was mixed in DMEM medium and allowed to stand at room temperature for 20 minutes.
- rAAV viruses carrying the full-length AFP cDNA of the tumor-associated antigen gene AFP gene or a part of the AFP cDNA fragment were named rAAV/AFP, r AAV/AmAFP, rAAV/BmAFP, rAAV/, respectively. CmAFP.
- the virus titer was determined by routine dot blot hybridization of various rAAV viruses (rAAV/AFP, rAAV/AmAFP rAAV/BmAFP, rAAV/CmAFP) obtained in step 1.
- the specific method includes the following steps: Only the DNA probe used A specific probe for a tumor-associated antigen gene.
- the nylon membrane was placed in a dot blotter, and the alkali-denatured rAAV virion DNA was added, and the DNA copy number standard was added, and vacuum was applied.
- Example 8-1 Step C.
- PCR DIG-labeled specific probe using the PCR DIG Labeling Kit and refer to the kit instructions.
- the probe is the AFP cDNA obtained in Example 8-1, Step C.
- PCR amplification After PCR amplification, PCR amplification The product was subjected to 1.2% agarose gel electrophoresis, and the PCR amplification product was detected under ultraviolet light, and a positive band appeared, indicating that the probe was successfully labeled.
- rMV/AFP the detection results of rMV/AFP are shown in Fig. 4H, the virus titer of rMV/AFP is ⁇ - ⁇ 11 copies/ml, and the virus titers of the three rAAV/AraAFP are 10'°-10' 1 copies/ mL.
- Example 8-3 Tumor-associated antigen-introduced monocyte-macrophage-dendritic cell line killing tumor Test
- rAAV virus rAAV/AFP and rMV/mAFP (rMV/AmAFP, rAAV/BmAFP, rAAV/CmAFP).
- AB1-V Cell Culture Medium purchased from Invitrogen, USA.
- Cytokines Colony stimulating factor (GM-CSF), interleukin 2, 4, 7 (IL-2, 4, 7) and tumor necrosis factor (TNF- ⁇ ) were purchased from R&D, USA.
- GM-CSF Colony stimulating factor
- IL-2 interleukin 2, 4, 7
- TNF- ⁇ tumor necrosis factor
- the whole process of killing a tumor-based tumor-inhibiting tumor of a tumor patient with one or more rAAV viruses carrying a tumor-associated antigen gene includes the following steps. :
- peripheral blood cell separator or lymphocyte separation solution
- PBMC peripheral blood mononuclear cells
- AIM-V medium remove suspended cells and retain adherent cells (monocytes, monocytes, Mo). Suspension cells, which are peripheral blood lymphocytes, are mixed with AIM-V medium and cultured for further use.
- the r V virus obtained in the embodiment 8-2 of the invention is added in an amount of about 100-1000 MOI, and then GM-CSF (800 IU/mL) is added, and the culture is continued. hour.
- the old medium was removed, and the AIM-V medium containing GM-CSF, IL-4 (800 IU/mL) and TNF-a (20 IU/mL) was added, and the culture was continued.
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- DC dendritic cells
- CTL cytotoxic T lymphocytes
- the conventional fluorescent antibody-labeled staining method was used to label the mononuclear cells or immature DCs infected with the rAAV of the present invention obtained by the specific fluorescent antibody against the tumor-associated antigen AFP (purchased from BD, USA), and then flow.
- the number of positive cells was measured by a cytometer.
- the efficiency of detection of peripheral blood mononuclear cells by recombinant adeno-associated virus rAAV/AFP is shown in Fig. 6H, and the efficiency of r V/AFP infection of peripheral blood mononuclear cells is 89%, and various tumor-bearing tumors constructed and prepared are constructed.
- the relative antigen gene rAAV (rAAV/AmAFP, rAAV/BmAFP, rAAV/CmAFP) is infected with peripheral blood mononuclear cells with an efficiency of about 90%, ie about 90% of peripheral blood mononuclear cells can be infected with rMV virus. It is proved that the rAAV of the present invention has high infection efficiency.
- DC dendritic cells
- the levels of DC expression CD80, CD83 and CD86 were positively correlated with the function of DC.
- the fluorescently labeled antibodies against these three CD molecules purchased from BD, USA
- Stimulated DCs and non-stimulated DCs were controls.
- the results of detection of CD80, CD83 and CD86 levels of DCs infected with recombinant adeno-associated virus rAAV/AFP are shown in Fig. 7H.
- r V/AFP and other rAAV carrying tumor-associated antigen gene-expressed DCs expressed significantly higher levels of CD molecules than controls, demonstrating the construction and preparation of tumor-associated antigen AFP After the rAAV of its mutant gene is infected with peripheral blood mononuclear cells, the induced DC is functional.
- CTL C cytotoxic T lymphocytes
- the function of CTL and its ability to kill tumor cells is positively correlated with the expression level of IFN- ⁇ .
- the level of CTL-expressing IFN-y induced by DCs infected with the rAAV of the present invention was detected by a method similar to that of Step A (CTL induced by non-stimulated DCs as a control), and DC and peripheral blood lymphocytes were mixed and cultured.
- the cells were harvested and labeled with fluorescent staining by conventional intracellular staining.
- the antibody used was a fluorescently labeled anti-antibody against IFN- ⁇ (purchased from BD, USA), and finally the results were detected by flow cytometry.
- the IFN- ⁇ expression level of CTL induced by rMV/AFP-infected DC is shown in Figure 8H, rAAV/AFP and other rAAV (rAAV/AmAFP, rAAV/BmAFP, rAAV/CmAFP) carrying tumor-associated antigen genes.
- the level of CTL expression of IFN- ⁇ induced by the infected DC was significantly higher than that of the control, demonstrating that the CTL induced by the rAAV-infected DC carrying the tumor-associated antigen AFP and its mutant gene constructed and prepared by the present invention is powerful.
- CTL Cytotoxicity T lymphocyte
- the cytotoxic T lymphocytes induced by DCs infected with rAAV were 20:1 (lymphocytes: tumor cells) and after the liver cells were mixed, using conventional methods and ⁇ 5 'Cr (chromium-51) anti-testing, testing the activity of CTL killing of tumor cells.
- the statistical results of tumor cell killing rate of CTL induced by rMV/AFP-infected DC are shown in Fig. 9H, and the CTL induced by the DC of the invention carrying and preparing the tumor-associated antigen AFP and its mutant gene can be more Effectively lyse (kill) tumor cells, the kill rate can reach more than 50%.
- rAAV cytotoxic T lymphocytes induced by infected DCs to kill tumor cells.
- Fig. 9H the tumor cell killing specificity test results of CTL induced by DCs infected with rAAV/AFP are shown in Fig. 9H, and the DCs carrying the tumor-associated antigen-AFP and its mutant rAAV infection constructed and prepared by the present invention are shown in Fig. 9H.
- the induced CTL has no killing effect on AFP antigen-negative cancer cells, and it is proved that the CTL induced by the rAAV-infected DC carrying the tumor-associated antigen-AFP and its mutant gene constructed and prepared according to the present invention has antigen specificity, that is, antigen-negative No killing of cells Function.
- Recombinant adeno-associated virus-dendritic cell technology which is to be infected with one or both of the inventions r V ( r AAV/AFP, rAAV/AmAFP, rAAV/BmAFP and rAAV/CraAFP) in Example 8-3
- the DCs induced by DC (rAAV-DC) were returned to 8 patients with liver cancer, and the infusion volume was 1 X 10 9 -5 X 10 9 .
- Treatment course usually 6 months, 2-3 times a month, after the condition is improved, it can be reduced to 1-2 times per month, and further reduced to once every 1-3 months.
- Therapeutic effect reaction after rAAV-DC treatment
- the statistical results are shown in Table 8-1 (B: serum tumor markers decreased or disappeared.
- rAAV-DC CTL induced by the rAAV-infected DC of the present invention
- rAAV-DC Recombinant adeno-associated virus-dendritic cell technology
- Clinical rMV carries the current diagnosis after treatment (month) treatment effect
- the imaging changes of the metastatic lesions before and after treatment in 8 patients with liver cancer in step 1 were performed.
- the results were obtained by rAAV (rAAV/LMP-U rAAV/AmLMP-1, rAAV/BmLMP-1 and rMV/CmLMP-1) of the present invention.
- rAAV-DC After treatment with CTL induced by one or two infected DCs (rAAV-DC), the metastatic lesion of the patient disappeared significantly, further demonstrating that CTL induced by the DCA-infected DC of the present invention can be effectively inhibited in the patient.
- the growth of malignant tumor cells or the killing of tumor cells can be used to prepare antitumor drugs.
- the levels of serum tumor-associated antigen AFP in the serum of 8 patients with liver cancer before and after treatment in step 1 were detected (data from the results of laboratory tests). Among them, the changes of serum AFP tumor-associated antigen levels in liver cancer patients before and after CTL treatment induced by rAAV/AFP-infected DCs are shown in Fig. 11H, and the results were obtained by the present invention rAAV (rAAV/AFP, rAAV/AmAFP, rAAV).
- the viral vector or the product associated with the recombinant adeno-associated virus vector of the present invention can be used for the preparation of an antitumor drug, in malignant tumors such as prostate cancer, epithelial cell malignancy, breast cancer, colon cancer, gastric cancer, lung adenocarcinoma, lung cancer, ovary It is of great significance in the clinical treatment and application of cancer, nasopharyngeal carcinoma, cervical cancer, lung squamous cell carcinoma and liver cancer.
Abstract
Cette invention a trait à des vecteurs recombinés dérivés du virus adéno-associé (RAAV), à leurs procédés de construction et leurs utilisations. Chacun des RAAV est obtenu en remplaçant un gène structurel du virus adéno-associé par l'un des gènes antigéniques tumoraux suivants : PSA, PSMA, CEA, CK19, Her-2/neu, LMP-1, BA46 ou AFP, ou par le type mutant de chaque gène, respectivement. Chacun des RAAV de la présente invention peut introduire le gène antigénique spécifique de la prostate qu'il porte, du type sauvage ou mutant, dans des lignées de monocytes-macrophages-cellules dendritiques pour stimuler les cellules effectrices du système immunitaire. Des données expérimentales ont montré que les lymphocytes T cytotoxiques induits par les cellules dendritiques, qui sont infectées avec l'un des RAAV de la présente invention, peuvent inhiber avec efficacité la croissance des cellules tumorales malignes ou éliminer les cellules tumorales in vivo chez le patient. Les vecteurs recombinés dérivés du virus adéno-associé de l'invention et leurs produits associés peuvent être utilisés pour préparer les médicaments utilisés dans le traitement du cancer de la prostate, des tumeurs malignes du tissu épithélial, du cancer du sein, du cancer du colon, du cancer de l'estomac, de l'adénocarcinome pulmonaire, du cancer du poumon, de l'oophorome, de l'épithélioma du rhinopharynx, du cancer du col de l'utérus, de l'épithélioma spinocellulaire du poumon, du cancer du foie, etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008800129496A CN101680002B (zh) | 2007-04-23 | 2008-04-23 | 一组重组腺相关病毒载体及其构建方法与应用 |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710097935 | 2007-04-23 | ||
| CN200710097935.6 | 2007-04-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008128440A1 true WO2008128440A1 (fr) | 2008-10-30 |
Family
ID=39875079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2008/000835 WO2008128440A1 (fr) | 2007-04-23 | 2008-04-23 | Série de vecteurs recombinés dérivés du virus adéno-associé (raav), leurs procédés de construction et leurs utilisations |
Country Status (2)
| Country | Link |
|---|---|
| CN (16) | CN102268456B (fr) |
| WO (1) | WO2008128440A1 (fr) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775375B (zh) * | 2009-07-31 | 2013-04-10 | 华中科技大学同济医学院附属同济医院 | 制备含eb病毒潜伏膜蛋白1,2基因的重组腺相关病毒及其应用 |
| CN108546715A (zh) * | 2018-01-19 | 2018-09-18 | 广东拓谱康生物科技有限公司 | 一种lmp-2重组腺相关病毒载体及其构建方法与应用 |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638655B (zh) * | 2009-09-03 | 2011-05-04 | 杭州安倍生物科技有限公司 | 癌胚抗原阳性细胞靶向性基因表达元件cpe及其应用 |
| CN105316361B (zh) * | 2014-08-01 | 2017-11-28 | 广东拓谱康生物科技有限公司 | 携带人乳头瘤病毒16型突变型E7m58抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN105177047B (zh) * | 2014-08-12 | 2018-02-02 | 广东拓谱康生物科技有限公司 | 携带人乳头瘤病毒16型突变型E7m94抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN105177048B (zh) * | 2014-08-12 | 2018-02-02 | 广东拓谱康生物科技有限公司 | 携带人乳头瘤病毒16型多点突变型E7mm抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN105018525B (zh) * | 2014-08-12 | 2017-06-13 | 广东拓谱康生物科技有限公司 | 携带人乳头瘤病毒16型突变型E7m91抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN104830780A (zh) * | 2015-05-05 | 2015-08-12 | 杨光华 | 基于ba46抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途 |
| CN104830782A (zh) * | 2015-05-05 | 2015-08-12 | 杨光华 | 基于ck19抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途 |
| CN104830800A (zh) * | 2015-05-05 | 2015-08-12 | 杨光华 | 基于psma抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途 |
| CN105087649B (zh) * | 2015-06-17 | 2018-10-02 | 深圳益世康宁生物科技有限公司 | 携带muc-1抗原基因的重组腺相关病毒载体及构建方法与应用 |
| CN105985984B (zh) * | 2015-06-17 | 2018-10-02 | 深圳益世康宁生物科技有限公司 | 携带pap抗原基因的重组腺相关病毒载体及构建方法与应用 |
| CN105087647B (zh) * | 2015-06-17 | 2018-10-02 | 深圳益世康宁生物科技有限公司 | 一种携带Survivin抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN105969804B (zh) * | 2015-06-17 | 2018-10-02 | 深圳益世康宁生物科技有限公司 | 一种携带scc抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN105087648B (zh) * | 2015-06-17 | 2018-05-25 | 深圳益世康宁生物科技有限公司 | 携带mage-a3抗原基因的重组腺相关病毒载体及构建方法与应用 |
| CN106591370A (zh) * | 2015-10-19 | 2017-04-26 | 南京华贞生物医药科技有限公司 | 一种治疗自身免疫性相关疾病以及糖尿病的病毒载体及其构建方法和应用 |
| CN106119231A (zh) * | 2016-06-24 | 2016-11-16 | 安徽未名细胞治疗有限公司 | 一种肿瘤抗原psa的ctl识别表位肽及其应用 |
| CN106282234B (zh) * | 2016-08-05 | 2020-11-13 | 深圳前海美康医疗生物技术有限公司 | 携带人c基因型乙型肝炎病毒的表面抗原s基因的重组腺相关病毒载体及其构建方法与应用 |
| CN108753825A (zh) * | 2018-06-11 | 2018-11-06 | 北京安斯晨睿生物科技有限公司 | 携带her2/erbb突变抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN108841867A (zh) * | 2018-06-11 | 2018-11-20 | 北京安斯晨睿生物科技有限公司 | 携带 scc/bst2 突变抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN108642085A (zh) * | 2018-06-11 | 2018-10-12 | 北京安斯晨睿生物科技有限公司 | 携带bcma突变抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN108893490A (zh) * | 2018-07-25 | 2018-11-27 | 北京安斯晨睿生物科技有限公司 | 携带ceacam8突变抗原基因的重组腺相关病毒载体及其构建方法与应用 |
| CN109234314B (zh) * | 2018-10-16 | 2022-06-07 | 汉恒生物科技（上海）有限公司 | 用于敲除cxcl12基因的腺相关病毒重组载体及其构建方法和用途 |
| CN110684800B (zh) * | 2018-11-02 | 2020-11-03 | 深圳益世康宁生物科技有限公司 | 一种携带肿瘤-睾丸抗原10基因的重组腺相关病毒载体及其应用价值 |
| CN110885855A (zh) * | 2018-11-02 | 2020-03-17 | 深圳益世康宁生物科技有限公司 | 一种携带精子蛋白17抗原基因的重组腺相关病毒载体及其应用价值 |
| JP7315965B2 (ja) * | 2018-11-09 | 2023-07-27 | 積水メディカル株式会社 | ウィルス性肝癌の検出方法 |
| CN111647606B (zh) * | 2020-08-06 | 2020-11-27 | 北京翊博普惠生物科技发展有限公司 | 靶向afp全抗原的dc细胞、ctl细胞及其制备方法和应用 |
| AU2021360902A1 (en) | 2020-10-15 | 2023-04-27 | Aavocyte, Inc. | Recombinant adeno-associated virus vectors with cd14 promoter and use thereof |
| CN116355058A (zh) * | 2020-11-12 | 2023-06-30 | 天津大学 | Eb病毒抗原表位及其应用 |
| CN113416729B (zh) * | 2021-05-18 | 2022-11-22 | 遵义医科大学附属医院 | 一种肝脏靶向调控甲胎蛋白基因的shRNA和cDNA及其应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6652850B1 (en) * | 1993-09-13 | 2003-11-25 | Aventis Pharmaceuticals Inc. | Adeno-associated viral liposomes and their use in transfecting dendritic cells to stimulate specific immunity |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5173414A (en) * | 1990-10-30 | 1992-12-22 | Applied Immune Sciences, Inc. | Production of recombinant adeno-associated virus vectors |
| US6254862B1 (en) * | 1997-03-03 | 2001-07-03 | Calydon, Inc. | Adenovirus vectors specific for cells expressing alpha-fetoprotein and methods of use thereof |
| CN100402655C (zh) * | 2005-03-17 | 2008-07-16 | 中山大学中山医学院科技开发中心 | 人神经营养素-3受体基因重组腺病毒构建方法 |
-
2008
- 2008-04-23 CN CN2011101256609A patent/CN102268456B/zh active Active
- 2008-04-23 CN CNA2008100932265A patent/CN101255442A/zh not_active Withdrawn
- 2008-04-23 CN CNA2008100932250A patent/CN101255441A/zh not_active Withdrawn
- 2008-04-23 CN CN201110125683XA patent/CN102268457B/zh active Active
- 2008-04-23 CN CNA2008100932195A patent/CN101307325A/zh not_active Withdrawn
- 2008-04-23 CN CN2011101256435A patent/CN102268455B/zh active Active
- 2008-04-23 WO PCT/CN2008/000835 patent/WO2008128440A1/fr active Application Filing
- 2008-04-23 CN CN2011101256030A patent/CN102277384B/zh active Active
- 2008-04-23 CN CNA2008100932208A patent/CN101307326A/zh not_active Withdrawn
- 2008-04-23 CN CN2008800129496A patent/CN101680002B/zh active Active
- 2008-04-23 CN CN2011101256651A patent/CN102268453B/zh active Active
- 2008-04-23 CN CNA2008100932246A patent/CN101307330A/zh not_active Withdrawn
- 2008-04-23 CN CN2011101256327A patent/CN102268454B/zh active Active
- 2008-04-23 CN CNA2008100932212A patent/CN101307327A/zh not_active Withdrawn
- 2008-04-23 CN CN2011101256971A patent/CN102268458B/zh active Active
- 2008-04-23 CN CNA2008100932231A patent/CN101307329A/zh not_active Withdrawn
- 2008-04-23 CN CNA2008100932227A patent/CN101307328A/zh not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6652850B1 (en) * | 1993-09-13 | 2003-11-25 | Aventis Pharmaceuticals Inc. | Adeno-associated viral liposomes and their use in transfecting dendritic cells to stimulate specific immunity |
Non-Patent Citations (4)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775375B (zh) * | 2009-07-31 | 2013-04-10 | 华中科技大学同济医学院附属同济医院 | 制备含eb病毒潜伏膜蛋白1,2基因的重组腺相关病毒及其应用 |
| CN108546715A (zh) * | 2018-01-19 | 2018-09-18 | 广东拓谱康生物科技有限公司 | 一种lmp-2重组腺相关病毒载体及其构建方法与应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102268456B (zh) | 2013-02-06 |
| CN101307329A (zh) | 2008-11-19 |
| CN102268456A (zh) | 2011-12-07 |
| CN102268457B (zh) | 2013-06-26 |
| CN101307330A (zh) | 2008-11-19 |
| CN101255442A (zh) | 2008-09-03 |
| CN102268455B (zh) | 2013-09-18 |
| CN102268453A (zh) | 2011-12-07 |
| CN101680002A (zh) | 2010-03-24 |
| CN102268454A (zh) | 2011-12-07 |
| CN102268454B (zh) | 2013-07-24 |
| CN102268458A (zh) | 2011-12-07 |
| CN101680002B (zh) | 2011-11-23 |
| CN102268453B (zh) | 2013-04-03 |
| CN102277384B (zh) | 2013-06-26 |
| CN102268455A (zh) | 2011-12-07 |
| CN102268457A (zh) | 2011-12-07 |
| CN102268458B (zh) | 2013-04-03 |
| CN101307327A (zh) | 2008-11-19 |
| CN101255441A (zh) | 2008-09-03 |
| CN102277384A (zh) | 2011-12-14 |
| CN101307328A (zh) | 2008-11-19 |
| CN101307325A (zh) | 2008-11-19 |
| CN101307326A (zh) | 2008-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2008128440A1 (fr) | Série de vecteurs recombinés dérivés du virus adéno-associé (raav), leurs procédés de construction et leurs utilisations | |
| US20190000943A1 (en) | Aav6 vectors for immunotherapy | |
| CN105985984B (zh) | 携带pap抗原基因的重组腺相关病毒载体及构建方法与应用 | |
| WO2016015684A1 (fr) | Vecteur recombinant basé sur le virus adéno-associé comprenant un gène muté codant pour l'antigène e7 du papillomavirus humain de type 16, méthode de construction de celui-ci, et application de celui-ci | |
| CN105087647B (zh) | 一种携带Survivin抗原基因的重组腺相关病毒载体及其构建方法与应用 | |
| CN105087648B (zh) | 携带mage-a3抗原基因的重组腺相关病毒载体及构建方法与应用 | |
| CN105018525B (zh) | 携带人乳头瘤病毒16型突变型E7m91抗原基因的重组腺相关病毒载体及其构建方法与应用 | |
| CN105177048B (zh) | 携带人乳头瘤病毒16型多点突变型E7mm抗原基因的重组腺相关病毒载体及其构建方法与应用 | |
| CN108546715A (zh) | 一种lmp-2重组腺相关病毒载体及其构建方法与应用 | |
| CN105969804B (zh) | 一种携带scc抗原基因的重组腺相关病毒载体及其构建方法与应用 | |
| CN106282234B (zh) | 携带人c基因型乙型肝炎病毒的表面抗原s基因的重组腺相关病毒载体及其构建方法与应用 | |
| CN110684800B (zh) | 一种携带肿瘤-睾丸抗原10基因的重组腺相关病毒载体及其应用价值 | |
| CN105177047B (zh) | 携带人乳头瘤病毒16型突变型E7m94抗原基因的重组腺相关病毒载体及其构建方法与应用 | |
| Koppold et al. | Efficient gene transfer of CD40 ligand into ovarian carcinoma cells with a recombinant adeno-associated virus vector | |
| CN111088286B (zh) | 一种重组腺相关病毒载体的构建和制备及其应用价值 | |
| CN110885855A (zh) | 一种携带精子蛋白17抗原基因的重组腺相关病毒载体及其应用价值 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| WWE | Wipo information: entry into national phase |
Ref document number: 200880012949.6 Country of ref document: CN |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08748407 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 08748407 Country of ref document: EP Kind code of ref document: A1 |