CN104830781A - HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof - Google Patents
HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof Download PDFInfo
- Publication number
- CN104830781A CN104830781A CN201510225001.0A CN201510225001A CN104830781A CN 104830781 A CN104830781 A CN 104830781A CN 201510225001 A CN201510225001 A CN 201510225001A CN 104830781 A CN104830781 A CN 104830781A
- Authority
- CN
- China
- Prior art keywords
- cell
- optionally
- hsv
- volume
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108091007433 antigens Proteins 0.000 title claims abstract description 74
- 239000000427 antigen Substances 0.000 title claims abstract description 72
- 102000036639 antigens Human genes 0.000 title claims abstract description 70
- 210000004027 cell Anatomy 0.000 title claims abstract description 70
- 241000701074 Human alphaherpesvirus 2 Species 0.000 title claims abstract description 69
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 230000008685 targeting Effects 0.000 title abstract description 5
- 238000000034 method Methods 0.000 claims abstract description 67
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 32
- 230000004069 differentiation Effects 0.000 claims abstract description 27
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 27
- 238000011282 treatment Methods 0.000 claims abstract description 15
- 238000001890 transfection Methods 0.000 claims description 37
- 206010028980 Neoplasm Diseases 0.000 claims description 36
- 239000002299 complementary DNA Substances 0.000 claims description 33
- 239000013612 plasmid Substances 0.000 claims description 32
- 239000006143 cell culture medium Substances 0.000 claims description 31
- 230000004913 activation Effects 0.000 claims description 27
- 230000008569 process Effects 0.000 claims description 26
- 238000000338 in vitro Methods 0.000 claims description 25
- 230000001939 inductive effect Effects 0.000 claims description 24
- 239000012679 serum free medium Substances 0.000 claims description 24
- 238000013518 transcription Methods 0.000 claims description 20
- 230000035897 transcription Effects 0.000 claims description 20
- 102100039497 Choline transporter-like protein 3 Human genes 0.000 claims description 17
- 101000889279 Homo sapiens Choline transporter-like protein 3 Proteins 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 14
- 210000001616 monocyte Anatomy 0.000 claims description 14
- 210000002966 serum Anatomy 0.000 claims description 11
- 102100031699 Choline transporter-like protein 1 Human genes 0.000 claims description 10
- 101000940912 Homo sapiens Choline transporter-like protein 1 Proteins 0.000 claims description 10
- 230000008859 change Effects 0.000 claims description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 10
- 108091008146 restriction endonucleases Proteins 0.000 claims description 10
- 102100035954 Choline transporter-like protein 2 Human genes 0.000 claims description 9
- 229930182566 Gentamicin Natural products 0.000 claims description 9
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 9
- 101000948115 Homo sapiens Choline transporter-like protein 2 Proteins 0.000 claims description 9
- 229960002518 gentamicin Drugs 0.000 claims description 9
- 230000035800 maturation Effects 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 102100039496 Choline transporter-like protein 4 Human genes 0.000 claims description 6
- 101000889282 Homo sapiens Choline transporter-like protein 4 Proteins 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 238000012546 transfer Methods 0.000 claims description 6
- 230000000527 lymphocytic effect Effects 0.000 claims description 5
- 230000005611 electricity Effects 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 238000009792 diffusion process Methods 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 22
- 238000009169 immunotherapy Methods 0.000 abstract description 10
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 3
- 230000003013 cytotoxicity Effects 0.000 abstract description 3
- 210000005259 peripheral blood Anatomy 0.000 abstract description 3
- 239000011886 peripheral blood Substances 0.000 abstract description 3
- 230000009385 viral infection Effects 0.000 abstract description 3
- 230000000638 stimulation Effects 0.000 abstract description 2
- 210000005087 mononuclear cell Anatomy 0.000 abstract 3
- 210000004443 dendritic cell Anatomy 0.000 description 65
- 210000004405 cytokine-induced killer cell Anatomy 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 12
- 230000001225 therapeutic effect Effects 0.000 description 11
- 241000700605 Viruses Species 0.000 description 10
- 238000012258 culturing Methods 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 10
- 238000002659 cell therapy Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 210000004881 tumor cell Anatomy 0.000 description 8
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 230000006698 induction Effects 0.000 description 6
- 230000009885 systemic effect Effects 0.000 description 6
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 5
- 238000002512 chemotherapy Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 230000000699 topical effect Effects 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000022534 cell killing Effects 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000155 melt Substances 0.000 description 4
- 210000000822 natural killer cell Anatomy 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000001959 radiotherapy Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000001815 biotherapy Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000973 chemotherapeutic effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 102100028704 Acetyl-CoA acetyltransferase, cytosolic Human genes 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 208000029433 Herpesviridae infectious disease Diseases 0.000 description 1
- 101100536896 Homo sapiens ACAT2 gene Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 208000032376 Lung infection Diseases 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 235000003283 Pachira macrocarpa Nutrition 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 240000001085 Trapa natans Species 0.000 description 1
- 235000014364 Trapa natans Nutrition 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 239000012578 cell culture reagent Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 231100001157 chemotherapeutic toxicity Toxicity 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical class ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 231100000284 endotoxic Toxicity 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000037850 immune anergy Diseases 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002647 laser therapy Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 238000000554 physical therapy Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000009165 saligot Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000000015 thermotherapy Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a DC cell and a targeting immune cell population, and a preparation method and application thereof. The preparation method of DC cells includes the steps of: conducting first induced differentiation culture on mononuclear cells in order to obtain immature DC cells; transfecting the immature DC cells by using HSV-2 antigen mRNA, so as to obtain transfected immature DC cells; and conducting second induced differentiation culture on the transfected immature DC cells, so as to obtain mature DC cells, wherein the mononuclear cells are isolated from mononuclear cells of peripheral blood of a sample. The method can prepare DC cells safely and efficiently, has a low cost and high efficiency, and can effectively solve the problem of cytotoxicity. The method can be effectively used for immune cell treatment or stimulation of lymphocytes derived from a same patient to produce a targeting cell population containing a large amount of CTL cells, and then the cell population can be used for virus infection immunotherapy to reach better effect.
Description
Technical field
The present invention relates to biological technical field, particularly, the present invention relates to DC cell, targeted immune cell mass and its production and use.
Background technology
Malignant tumour is the main fatal disease types of the mankind, and mortality ratio remains high.It is the first that Ministry of Health's recent statistics data show that malignant tumour occupies China resident top ten cause of the death.Chinese Incidence patient 2,680,000 people in 2010, dead 1,970,000 people; The current whole nation has malignant tumor patient more than 600 ten thousand people.Medical expense every year for malignant tumor patient accounts for 20% of Health Expenditure, is the maximum medical burden of the whole society.Current treatment means for reaching healing, to extend the effect of malignant tumor patient lifetime and the targets such as quality of making the life better still very limited, even the malignant tumour early diagnosis especially for some type is also difficult to obtain satisfied curative effect, the malignant tumour as transitivity, general or progressive stage is then more difficult to cure.
In current ideas of cancer therapy, the topical therapeutics such as operation and radiotherapy have good curative effect to limitation tumour, then mainly rely on chemotherapy or immunotherapy to carry out systemic treatment for the MRD after general, transitivity or topical therapeutic.Tumor invasion is the result of multi-step, polygene effect, and tumour cell shows growth, differentiation and apoptosis out of control.Clinically the diversity of patient symptom and idiogenetics heterogeneous, and the appearance of tumour distant metastasis focus, makes oncotherapy with the viewpoint of systemic disease, must adopt the comprehensive therapeutic plan of individuation.Namely according to the feature of each tumour patient, adopt topical therapeutic in conjunction with whole body therapeutic; Not only to eliminate the tumour of local lesion, also will control the Preventive growth of tumour and tumour to the invasion and attack of important organ, really effectively could improve curative ratio and the lifetime of tumour patient.The treatment of usual tumor by local focus can take operation and Physiotherapy (comprise radio-frequency (RF) ablation, thermotherapy, radiotherapy, freezing, ultrasonicly involve laser therapy etc.) method elimination, but for the not detectable tumour minimal disease of iconography (as the residual after transfer and relapse stove, topical therapeutic, the cancer cells in blood), above topical therapeutic means are then helpless, must be solved by systemic treatment means.Because the transfer of tumour, recurrence, diffusion are more fatal, therefore systemic treatment seems particularly important and urgent.Current systemic treatment has the treatment of traditional chemotherapy, molecular targeted agents, biotherapy (comprising cellular replacement therapy and immune cell therapy) and gene therapy etc.Although classic chemotherapy has made some progress in some oncotherapy, prolongation tumour patient aspect lifetime is still contributed not quite.And tumour cell is to the natural drug resistance of chemotherapeutic and Acquired lung infection and chemotherapeutic toxicity, makes chemotherapeutical development receive serious obstruction.Clinical practice for many years proves that it is not the strong event of whole body therapeutic.
Immune cell therapy is that recent two decades grows up, and immune cell therapy mainly comprises CIK and DC cell.Due to its therapeutic domain wide (can be used for noumenal tumour and leukemia), more effective to tumour minimal disease (comprising the cancer cells in transfer, recurrence stove, blood) especially, have no side effect, and be applicable to tumour each stage (as Radiotherapy chemotherapy in late period can not use), and be equally applicable to the tumour that virus infection causes; Therefore it is a strong event of whole body therapeutic.Immune cell therapy has significant effect to raising quality of life, prolongation lifetime, and praise highly extremely both at home and abroad, be one of best means of current whole body therapeutic, clinical potentials is huge.
But the preparation method at present for the immunocyte carrying out immune cell therapy still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind of safe, efficient method preparing DC cell and targeted immune cell mass.
It should be noted that, the present invention completes based on the following discovery of contriver:
Along with the development of biotherapy technology, immunotherapy is carried out to malignant tumour and becomes study hotspot, immunotherapy is the brand-new pattern of one of oncotherapy, and especially cell-mediated adoptive immunotherapy comparatively maturation has entered the clinical application stage, and achieves obvious result for the treatment of.Cell-mediated adoptive immunotherapy has become one of important means of assisting therapy after tumour patient Radiotherapy chemotherapy, its for promoting reconstruction of patients immune system, eliminate residual and bone marrow purging all has good result.People kill and wound (LAK) cell, tumor-infiltrated lymph (TIL) cell, cytokine-induced killer cell (CIK) cell etc. by research lymphokine-activation, show CIK cell be a kind of novel, efficient, there is non-principal histocompatibility complex (MHC) the restricted immune effector cell that wide spectrum kills tumor activity, in immunotherapy of tumors, manifest huge using value.Dendritic cell (DC) is main antigen presenting cell powerful in human body, antigen-specific cellular poison lymphocyte (CTL) can be induced react and the propagation affecting β cell (cell of Regular Insulin can be produced in the pancreas islet of pancreas) by direct or indirect mode, activation humoral immunoresponse(HI).By CIK cell and DC combination therapy malignant tumour, contribute to the T cell Immune anergy removing tumour patient, have the effect of synergistic antitumor.The treatment (CIK/DC cell therapy) of cytokine induced kill cell associating dendritic cell to malignant tumour that increasing test and clinical practice show demonstrates good therapeutic action, has huge development potentiality and application prospect.
Wherein, CTL cell is the T cell subgroup of CD8+, is a kind of specific T cell, has direct killing effect to some virus, tumour cell etc., forms body disease-resistant poison, antineoplastic important defence line with natural killer cell.CTL kill mechanism has: 1. discharge pore-forming protein, granzyme, cracking target cell.2. the apoptosis of target cell is mediated by FasL.
Antigen load technology main Types based on DC has: tumor antigen peptide impacts DC; Tumour cell antigen impacts DC; Tumour cell RNA impacts DC; The immunotherapy of genetic modification DC.Contriver finds; tumor-antigen peptide load DC is that application tumor-antigen peptide impacts sensitization DC in vitro; then feedback or immunization are to lotus knurl host; body can be obviously induced to produce Peptide-specific CTL; produce protective immunological reaction; dc Antigens all is at present developed as vaccine, produces antibody and CTL in body, does not prepare CTL cell in vitro.The immune response evoked in vivo due to antigen likely produces the possible of systemic font immunity, and contriver utilizes this technology to prepare CTL cell and combined immune cell mass in vitro, can solve current problem.In addition, current useful various carrier is as antigen, the viral vector Antigens such as rAAv prepare CTL technology, ACTL technology is exactly one of them, but due to the integration characteristic of virus, cause the genome toxicity problem of DC cell to can't resolve always, so contriver utilizes the carrier of mRNA antigen technical substitution virus type or DNA class, viral vector has been exceeded from security, simultaneously industrialization angle, mRNA in-vitro transcription more can realize industrialization, simple to operate, and Quality Control and quality inspection can stdn.
In other words, the present invention utilizes mRNA-DC-CTL technology to prepare targeted immune cell mass, particularly: by the peripheral blood lymphocytes (Monocytes) of the mRNA transfection patient of tumour/VAA determinant, through cytokine induction, monocyte transformation is the DC cell with powerful antigen presentation function.By the DC cell obtained through this technology, stimulate the T lymphocyte of patient in vitro, produce the cytotoxic T lymphocyte (CytotoxicTlymphocytes, CTL) of effective killing tumor cell/virus.The CTL produced has tumour/virus antigen specificity, i.e. targeting, and the tumour cell/virus only for certain or several specific tumors/VAA positive has lethal effect, to the cell of antigen negative without any effect.
And then, according to an aspect of the present invention, the invention provides a kind of method preparing DC cell.According to embodiments of the invention, the method comprises the following steps: monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell; Utilize immature DC cell described in HSV-2 antigen mRNA transfection, to obtain the immature DC cell through transfection; And the described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell, wherein, described monocyte is separated to obtain from the peripheral blood mononuclear cell of sample.
Contriver is surprised to find, utilize method of the present invention can prepare DC cell (i.e. dendritic cell) safely and efficiently, and method cost of the present invention is low, efficiency is high and can effectively solve cytotoxicity problem, safety non-toxic after the DC cell feedback patient obtained, immune cell therapy can be effective to, and the lymphocyte that this DC cell can also be effective to stimulate same patient to originate produces the targeted immune cell mass being rich in a large amount of CTL cell, and then for better effects if after the immunotherapy of tumour and virus infection.
In addition, the method for the DC of preparation cell according to the above embodiment of the present invention can also have following additional technical characteristic:
According to embodiments of the invention, described HSV-2 antigen mRNA is obtained by following steps: preparation HSV-2 antigen cDNA plasmid, and the nucleotide sequence of described HSV-2 antigen cDNA is as shown in SEQ ID NO:1; Described HSV-2 antigen cDNA plasmid is carried out linearization process, to obtain the HSV-2 antigen cDNA plasmid through linearization process; And the described HSV-2 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain HSV-2 antigen mRNA.Thereby, it is possible to effectively prepare HSV-2 antigen mRNA.
According to embodiments of the invention, with pcDNA3.1 carrier for skeleton carrier, prepare described HSV-2 antigen cDNA plasmid.Thus, preparation efficiency is high, effective.
According to embodiments of the invention, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, carry out described linearization process based on restriction enzyme site Bst1107I.
According to embodiments of the invention, T7Message Machine test kit is utilized to carry out described in-vitro transcription.Thus, transcriptional efficiency is high, effective.
According to embodiments of the invention, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.Thus, the carrying out of follow-up HSV-2 antigen mRNA transfection is conducive to.
According to embodiments of the invention, utilization electricity turns or PEI method carries out described transfection.Thus, transfection efficiency is high, effective.
According to embodiments of the invention, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described HSV-2 antigen mRNA.Thus, transfection is given prominence to.
According to embodiments of the invention, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation.Thereby, it is possible to effectively induction obtains DC cell.
According to embodiments of the invention, described DC substratum comprises: serum-free cell culture medium; The CTL4 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, culture effect is given prominence to.
According to embodiments of the invention, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.According to embodiments of the invention, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.Thus, culture effect is good.
According to a further aspect in the invention, present invention also offers a kind of method preparing targeted immune cell mass.According to embodiments of the invention, the method comprises the following steps: the peripheral blood mononuclear cell providing sample; The peripheral blood mononuclear cell of described sample is separated into lymphocyte and monocyte; Described lymphocyte is carried out activation culture, to obtain the lymphocyte through activation culture; Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell; Utilize immature DC cell described in HSV-2 antigen mRNA transfection, to obtain the immature DC cell through transfection; The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell; And the DC cell of described maturation and the described lymphocyte through activation culture are carried out mixed culture, to obtain targeted immune cell mass.
Contriver is surprised to find, method of the present invention is utilized can effectively to prepare targeted immune cell mass, this targeted immune cell mass not only has the powerful CTL cell killing and wounding specific tumors cell/virus, also has the immunocytes such as CIK, NK, can be effective to immune cell therapy.In addition, method cost of the present invention is low, efficiency is high, and can effectively solve cytotoxicity problem, safety non-toxic after the targeted immune cell mass feedback patient of acquisition.
According to embodiments of the invention, described HSV-2 antigen mRNA is obtained by following steps: preparation HSV-2 antigen cDNA plasmid, and the nucleotide sequence of described HSV-2 antigen cDNA is as shown in SEQ ID NO:1; Described HSV-2 antigen cDNA plasmid is carried out linearization process, to obtain the HSV-2 antigen cDNA plasmid through linearization process; And the described HSV-2 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain HSV-2 antigen mRNA.Thereby, it is possible to effectively prepare HSV-2 antigen mRNA.
According to embodiments of the invention, with pcDNA3.1 carrier for skeleton carrier, prepare described HSV-2 antigen cDNA plasmid.Thus, preparation efficiency is high, effective.
According to embodiments of the invention, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, carry out described linearization process based on restriction enzyme site Bst1107I.
According to embodiments of the invention, T7Message Machine test kit is utilized to carry out described in-vitro transcription.Thereby, it is possible to effectively carry out plasmid clone and follow-up linearization process.
According to embodiments of the invention, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.Thus, the carrying out of follow-up HSV-2 antigen mRNA transfection is conducive to.
According to embodiments of the invention, utilization electricity turns or PEI method carries out described transfection.Thus, transfection efficiency is high, effective.
According to embodiments of the invention, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described HSV-2 antigen mRNA.Thus, transfection is given prominence to.
According to embodiments of the invention, described activation culture comprises: utilize CTL1 cell culture medium to carry out the first activation culture 24 hours to described lymphocyte; CTL2 cell culture medium is utilized to carry out the second activation culture 2-3 days to the lymphocyte through the first activation culture; And utilize CTL3 cell culture medium to carry out the 3rd activation culture 4-5 days to the lymphocyte through the second activation culture, every equivalent fluid infusion in 2-3 days once.Thereby, it is possible to effectively make lymphocyte activation, be conducive to the acquisition of follow-up targeted immune cell mass.
According to embodiments of the invention, described CTL1 cell culture medium comprises: serum-free cell culture medium;
The CTL1 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, described CTL2 cell culture medium comprises: serum-free cell culture medium; The CTL2 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, described CTL3 cell culture medium comprises: serum-free cell culture medium; The CTL3 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, activation culture is effective.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, activation culture is remarkably productive, and lymphocyte activation is effective, is conducive to the acquisition of follow-up targeted immune cell mass.
According to embodiments of the invention, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation.Thereby, it is possible to effectively induction obtains DC cell.
According to embodiments of the invention, described DC substratum comprises: serum-free cell culture medium; The CTL4 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thus, culture effect is given prominence to.
According to embodiments of the invention, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.According to embodiments of the invention, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.Thus, culture effect is good.
According to embodiments of the invention, CTL3 cell culture medium is utilized to carry out described mixed culture 6-8 days, preferably 7 days.Thereby, it is possible to effectively obtain targeted immune cell mass.
According to embodiments of the invention, described CTL3 cell culture medium comprises: serum-free cell culture medium; The CTL3 factor of 100 ~ 1000U/ml; And 2 autoserums of volume % ~ 10 volume %, wherein, described serum origin is in described sample.Thus, culture effect is good.According to embodiments of the invention, described serum free medium is the serum free medium of Takara.According to other embodiments of the present invention, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin.Thereby, it is possible to prepare targeted immune cell mass efficiently.
According to embodiments of the invention, be that 1:20 carries out described mixed culture according to the DC cell of described maturation and the described lymphocytic volume ratio through activation culture.Thus, mixed culture is effective, can prepare targeted immune cell mass efficiently.
In addition, according to other embodiments of the present invention, the method preparing targeted immune cell mass of the present invention can also comprise the following steps:
The first step: extract nerpes vinrus hominis's infected patient vein peripheral blood (50-100 milliliter) or adopt blood cell separator directly to isolate peripheral blood mononuclear cell (PBMC).
Second step: through cultivating, PBMC is separated into lymphocyte and monocyte, lymphocyte continues to cultivate, for subsequent use; Utilize HSV-2 antigen mRNA transfection monocyte, and utilize the dendritic cell (DC) of cytokine induction maturation to produce.
3rd step: by the DC cell of maturation and lymphocyte mixed culture, becomes cytotoxic T lymphocyte that have antigen-specific, that kill and wound antigen positive tumour cell/virus by lymphocyte induction, obtains targeted immune cell mass.
According to another aspect of the invention, present invention also offers a kind of targeted immune cell mass.According to embodiments of the invention, it is prepared by the foregoing method preparing targeted immune cell mass.Contriver finds, targeted immune cell mass of the present invention not only has the powerful CTL cell killing and wounding specific tumors cell, also have the immunocytes such as CIK, NK, and cost is low, safety non-toxic, can be effective to immune cell therapy.
In accordance with a further aspect of the present invention, the DC cell that present invention also offers targeted immune cell mass and utilize the method for the foregoing DC of preparation cell to prepare is preparing the purposes in medicine, described medicine is used for the treatment of HSV-2 to be infected, or suppresses the transfer of HSV-2 positive tumor, diffusion, recurrence.
According to embodiments of the invention, described HSV-2 infects for nerpes vinrus hominis infects.
It should be noted that, contriver finds, because mRNA is without the need to promotor, can not unconfinedly increase, can not enter on nucleus Insertion Into Host Cell karyomit(e), without hereditary genotoxicity, the features such as the security that tool is very high, thus the present invention intends preparing DC cell and targeted immune cell mass (the targeted immune cell cluster for HSV-2) by mRNA-DC-CTL method, for use in the immunotherapy that tumour and nerpes vinrus hominis infect.Method of the present invention breaches the medicine of stimulation DC in the past can not the limitation of conventional preparation.Further, utilize mRNA transfection DC in vitro and produce belonging to for the first time of specificity Mutiple Targets CTL cell.
Particularly, method of the present invention have the following advantages one of at least:
1, HSV-2mRNA antigen is constructed first in the world.
2, HSV-2 antigen mRNA transfection DC cell is utilized to carry out external evoked acquisition HSV-2 specific target immunocyte group (being rich in CTL cell) in the world first.
3, target immunocyte group of the present invention comprises: CTL cell (CD8+, CD3+CD8+CD28+), CIK cell (CD3+CD56+), NK cell (CD3-CD56+), make it not only have the powerful CTL cell killing and wounding specific virus, also have the immunocytes such as CIK, NK, the overall immunity of patient can be promoted, the obvious symptom improving patient, can help that immune response is more complete, usefulness is higher.
4, the whole process that prepared by target immunocyte group only needs 12-14 days.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows according to one embodiment of the invention, the result of plasmid enzyme restriction qualification in HSV-2 antigen cDNA plasmid construction process;
Fig. 2 shows according to one embodiment of the invention, the result figure of effect after GFP detection mRNA transfection;
Fig. 3 shows according to one embodiment of the invention, the statistic analysis result that the flow cytomery result based on targeted immune cell mass obtains.
Embodiment
It should be noted that, term " first ", " second " only for describing object, and can not be interpreted as instruction or hint relative importance or imply the quantity indicating indicated technical characteristic.Thus, be limited with " first ", the feature of " second " can express or impliedly comprise one or more these features.Further, in describing the invention, except as otherwise noted, the implication of " multiple " is two or more.
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be and by the conventional products of commercial acquisition, such as, can be able to purchase from Illumina company.
Embodiment 1:
One, material and equipment
1, laboratory rank: GLP
2, plant and instrument:
Negative 80 degree of Ultralow Temperature Freezers (the U.S. water chestnut of middle section, DW-HL388); General refrigerator (Haier, double door ,-20 DEG C and 4 DEG C); Mold incubator (Shanghai is won fast, MJX-160B-Z); Constant water bath box (SSW-420-2S); CO
2incubator (HF240); Low speed centrifuge (Town in Shanghai booth, TDL-40C); Inverted biologic microscope (section believes, model: BLD-1); Biohazard Safety Equipment (Suzhou purifies, double); Electric pipettor (German Pulan moral) 1-10ml; Micropipette rifle (big dragon) 1 overlaps, 4; Program temperature reduction box (Thermo); Liquid nitrogen container (Jin Feng, YDS-120-216); PCR instrument; Gel electrophoresis imaging system; Electrophoresis apparatus; Thermostat;
3, cell cultures consumptive material:
Cell culture bags (Takara company); 75cm
2culturing bottle, 175cm
2culturing bottle, 1.8ml cryopreservation tube, 1.5ml EP pipe, 50ml centrifuge tube, 15ml centrifuge tube, all from Coning company of the U.S.; 0.22um syringe needle filter (U.S. company BD); 100um cell screen cloth (U.S. company BD); 30ml syringe (Shanghai Medical apparatus).
4, cell culture reagent:
Serum-free cell culture medium (Takara, article No.: GT-T561); Australia's foetal calf serum (FBS QUALIFIEDAUSTRALIA ORIGIN), Gibco, article No.: 10099-141;
Lymphocyte separation medium Ficoll liquid (GE, article No.: 17-1440-02);
The dual anti-solution of penicillin/streptomycin (PENICILLIN STREPTOMYCIN SOL, PS), Gibco, article No.: 15140-122.
5, the preparation of substratum:
5.1 reagent:
The CTL1 factor (comprising IFN-γ, 1000U/ml): 1ml, preserves for-20 DEG C for a long time, preserves 2 weeks for 4 DEG C;
The CTL2 factor (comprises IL-1,1000U/ml; IL-2,1000U/ml; CD3 antibody, 100ng/ml; CD28 antibody, 100ng/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
The CTL3 factor (comprises IL-2,1000U/ml; IL-7,20ng/ml; IL-15,20ng/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
The CTL4 factor (comprises GM-CSF, 1000U/ml; IL-4,500U/ml; TNF-α, 500U/ml; IL-1,500U/ml; IL-6,500U/ml): 1ml, preserves for a long time for-20 DEG C, preserves 2 weeks for 4 DEG C;
Tumour antigen mRNA transfection mixture reagent A: 100ul, preserves for a long time, once uses up after dissolving for-80 DEG C.
5.2 compound methods:
Basic medium: add the gentamicin of 2% in serum-free cell culture medium, wherein use after 0.22um syringe needle frit.
CTL1 substratum: the 1 pipe CTL1 factor taken out from-20 degree, normal temperature melts, and is added in 20ml basic medium, and adds the serum of the client of 10%, wherein use after 0.22um syringe needle frit.
CTL2 substratum: the 1 pipe CTL2 factor taken out from-20 degree, normal temperature melts, and is added in 20ml basic medium, wherein uses after 0.22um syringe needle frit.
CTL3 substratum: the 1 pipe CTL3 factor taken out from-20 degree, normal temperature melts, and is added in 500ml basic medium, wherein uses after 0.22um syringe needle frit, whole culturing process 3 pipes.
DC substratum: the 1 pipe CTL4 factor taken out from-20 degree, normal temperature melts, and is added in 50ml basic medium, and adds the serum of the client of 10%, wherein use after 0.22um syringe needle frit.
Two, method
1 builds HSV-2 antigen cDNA plasmid
First, HSV-2 antigen gene is carried out synthetic, and obtain the cDNA of gene.
The cDNA sequence of HSV-2 antigen gene is as follows:
ATGGGGCGTTTGACCTCCGGCGTCGGGACGGCGGCCCTGCTAGTTGTCGCGGTGGGACTCCGCGTCGTCTGCGCCAAATACGCCTTAGCAGACCCCTCGCTTAAGATGGCCGATCCCAATCGATTTCGCGGGAAGAACCTTCCGGTTTTGGACCAGCTGACCGACCCCCCCGGGGTGAAGCGTGTTTACCACATTCAGCCGAGCCTGGAGGACCCGTTCCAGCCCCCCAGCATCCCGATCACTGTGTACTACGCAGTGCTGGAACGTGCCTGCCGCAGCGTGCTCCTACATGCCCCATCGGAGGCCCCCCAGATCGTGCGCGGGGCTTCGGACGAGGCCCGAAAGCACACGTACAACCTGACCATCGCCTGGTATCGCATGGGAGACAATTGCGCTATCCCCATCACGGTTATGGAATACACCGAGTGCCCCTACAACAAGTCGTTGGGGGTCTGCCCCATCCGAACGCAGCCCCGCTGGAGCTACTATGACAGCTTTAGCGCCGTCAGCGAGGATAACCTGGGATTCCTGATGCACGCCCCCGCCTTCGAGACCGCGGGTACGTACCTGCGGCTAGTGAAGATAAACGACTGGACGGAGATCACACAATTTATCCTGGAGCACCGGGCCCGCGCCTCCTGCAAGTACGCTCTCCCCCTGCGCATCCCCCCGGCAGCGTGCCTCACCTCGAAGGCCTACCAACAGGGCGTGACGGTCGACAGCATCGGGATGCTACCCCGCTTTATCCCCGAAAACCAGCGCACCGTCGCCCTATACAGCTTAAAAATCGCCGGGTGGCACGGCCCCAAGCCCCCGTACACCAGCACCCTGCTGCCGCCGGAGCTGTCCGACACCACCAACGCCACGCAACCCGAACTCGTTCCGGAAGACCCCGAGGACTCGGCCCTCTTAGAGGATCCCGCCGGGACGGTGTCTTCGCAGATCCCCCCAAACTGGCACATCCCGTCGATCCAGGACGTCGCGCCGCACCACGCCCCCGCCGCCCCCAGCAACCCGGGCCTGATCATCGGCGCGCTGGCCGGCAGTACCCTGGCGGTGCTGGTCATCGGCGGTATTGCGTTTTGGGTACGCCGCCGCGCTCAGATGGCCCCCAAGCGCCTACGTCTCCCCCACATCCGGGATGACGACGCGCCCCCCTCGCACCAGCCATTGTTTTACTAGA(SEQ ID NO：1)
Then, with pcDNA3.1 carrier for skeleton carrier, by enzymatic cleavage methods, the HSV-2 antigen gene cDNA sequence shown in the SEQ ID NO:1 of above-mentioned synthetic is cloned on carrier, prepares HSV-2 antigen cDNA plasmid.
Wherein, Fig. 1 is shown in by plasmid enzyme restriction picture.As shown in Figure 1, from left to right, leftmost side swimming lane is: DNA marker, from top to bottom, and band order: 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp, 5000bp, 10000bp; Two swimming lanes in right side are: A040-1 plasmid is by carrying out digestions acquisition to the BamHI/SalI restriction enzyme site of pcDNA3.1 carrier.
Cut qualification through order-checking and enzyme, the HSV-2 gene order of synthetic is correct, and contriver successfully builds and obtains HSV-2 antigen cDNA plasmid.
2 in-vitro transcription
2.1 linearization process
Get the HSV-2 antigen cDNA plasmid built, the linearizing restriction enzyme site Bst1107I (distance terminator codon is distant, about 2KB) based on the suggestion of pcDNA3.1 carrier carries out linearization process.
2.2 in-vitro transcription
Utilize T7Message Machine test kit (Ambion company), the HSV-2 antigen cDNA plasmid (namely as the DNA profiling of in-vitro transcription) through linearization process is carried out in-vitro transcription, specific as follows:
First, in the plastic centrifuge tube of a RNase-free, at room temperature add following ingredients in order, preparation feedback system (20 microlitre):
Then, wrap the mouth of pipe with sealed membrane, 60 DEG C of water-bath 30min, the RNase inhibition in reaction system is played one's part to the full, to remove the RNase in template.
1 microlitre t7 rna polymerase (note: this is the consumption of 20 microlitre reaction systems is added after the cooling of question response system, to other reaction systems, the consumption of each composition can increase and decrease in proportion), 37 DEG C of insulations 2 hours (noting: extending soaking time can not significantly improve output).
Then, 70 DEG C of heating, 10 minutes deactivation t7 rna polymerases.
2.3 remove DNA profiling
In above-mentioned in-vitro transcription system, add 1 microlitre RNase-free DNase (3-5U/uL), and be incubated 15-30 minute at 37 DEG C, then moisturizing is to 100uL;
Then, with the saturated phenol-chloroform extracting of isopyknic Tris once;
Next, add the ethanol of 3M NaAc and the 2 times volume of 0.1 times of volume, the centrifugal 15-30 minute of maximum speed after mixing, add the ethanol of 1mL 70% after abandoning supernatant, after vibration, centrifugal 2 minutes of maximum speed, abandons supernatant, dry, the RNA of gained precipitation and in-vitro transcription gained.
Get 1-3 microlitre product electrophoresis detection transcription effects, detect qualified after the RNA obtained is placed in-80 DEG C of preservations.
3 targeted immune cell mass preparations
3.1. the separation of peripheral blood mononuclear cell
The cell obtained is separated in the 100ml peripheral blood that simplexvirus patient from the 1 routine HSV-2 positive is gathered, transfer to centrifugal (1800rpm in 50ml centrifuge tube, 10min), suck supernatant, getting the liquid after supernatant is slowly added on lymphocyte separation medium Ficoll liquid (GE), volume ratio is 1:1, centrifugal (2000rpm, 20min);
Then, the white flock cell collecting interface adds PBS, blows and beats mixing gently, centrifugal (1500rpm, 10min);
Next, repeated centrifugation washing totally 3 times;
Then, collect cell and blow and beat mixing gently, add basic medium 20ml and blow and beat evenly, be transferred to 75cm
2in culturing bottle, in 37 DEG C, 5%CO
22h cultivated by incubator.
3.2 lymphocytes are separated with monocytic
Take out 75cm
2culturing bottle, gently by 75cm
2culturing bottle straightens vertical, draws 18ml nutrient solution in new 175cm
2in bottle, for use in lymphocytic activation culture;
For former bottle (75cm
2culturing bottle) in remaining 2ml raffinate, monocyte will be used for be induced to differentiate into DC cell.
3.3 lymphocytic cultivations
For cultivating lymphocytic 175cm
2in bottle, add 20ml CTL1 cell culture medium, after 24h, add 20mlCTL2 culture medium culturing again, then every 2 ~ 3d CTL3 substratum equivalent fluid infusion, whether qualifiedly carry out active survival rate test checking.
3.4. monocytic process
A, at the surplus former bottle (75cm having 2ml raffinate
2culturing bottle) in add 15ml DC substratum and continue cultivation 4 days, within every 2 ~ 3 days half, change liquid once, monocyte to be induced to differentiate into immature DC cell.
B, PEI infection protocol carries out antigen gene mRNA transfection: the 5th day, immature DC cell (centrifugal 1500rpm is collected with 50ml centrifuge tube, 10min), after discarding supernatant, lower confluent monolayer cells is transferred to 1.5ml centrifuge tube, then reagent A (namely HSV-2mRNA and PEI mixes the rear mixture obtained according to mass ratio 1:3) the fully mixing bottom cell of 100ul is directly added, build centrifuge tube, be placed in cell culture incubator and hatch 30 minutes, take out turned upside down several times every 10 minutes.
Wherein, PEI (1ug/ul) Polysciences (CAT#23966-2 is mixed with liquid storage) obtains according to following steps preparation: first, will be heated to about 80 DEG C dissolve PEI, cool to room temperature without endotoxic sterilized water; Then, adjusted to ph to 7.0, sterilizes with the frit of 0.22um, is stored in-20 DEG C after packing, and working fluid can save backup at 4 DEG C.
GFP detects the effect after mRNA transfection, and as shown in Figure 2, wherein, left figure is picture before DC transfection to result, and right figure is picture after DC transfection, and transfection efficiency is up to 80% as shown in Figure 2.
C, take out centrifuge tube afterwards, with liquid-transfering gun cell suspension taken out and be added to culturing bottle (75cm
2) in, and add DC substratum, continue cultivation 3 days, to obtain ripe DC cell.
3.5. Dual culture
After 8th day, the DC cell of maturation is mixed according to volume ratio 1:20 with the aforementioned lymphocyte through activation culture, with CTL3 substratum Dual culture seven days, the generation of induction CTL cell and homicidal wound sexual cell thereof.
Dual culture, after seven days, carries out flow cytometer detection to cell, and then convection type detected result carries out statistical study.Wherein, Fig. 3 shows the statistic analysis result that the flow cytomery result based on targeted immune cell mass obtains, the per-cent namely in targeted immune cell mass shared by various immunocyte.As shown in Figure 3, not only containing a large amount of CTL cells in the immunocyte group that the present embodiment prepares, and also have the panimmunity such as NK and CIK cell cell: CTL cell (CD8+, CD3+CD8+CD28+), CIK cell (CD3+CD56+), NK cell (CD3-CD56+), namely this immunocyte group not only has the powerful CTL cell killing and wounding specific tumors cell/virus, also has the immunocytes such as CIK, NK.
Thus, contriver finds, transfected dendritic cell in vitro can activated T lymphocytes effectively, produces CTL, and enormous amount, reaches 10
9more than individual.Guarantee clinical efficacy is good.And traditional DC cell attempts activated T lymphocytes in vivo, produce CTL, but the immunity system microenvironment of the weakness of patient makes DC cell be difficult to activated T cell generation CTL in vivo, quantity is few, causes unsatisfactory curative effect, and without specificity, targeting.
Embodiment 2: clinical treatment
CTL cell embodiment 1 prepared feeds back patient, and observes feedback effect, specific as follows:
The simplexvirus patient 20 of the HSV-2 positive is divided into 2 groups (A group 10 and B groups 10), wherein, for A group, according to the method in embodiment 1 after the 14th day and the 15th day obtain enough CTL cells, feed back to patient, each feedback cell concentration is 1 ~ 2*10
9; B group does not feed back cell (in contrast).It is for 2 times a course for the treatment of that blood sampling one-time continuous feeds back.After 3 courses for the treatment of terminate, observe.
Found that: A group and B group have obvious difference, all patients of A group all feel that after feedback health sense tired out alleviates, and appetite increases, pain relief, and body weight increases, and wherein has 6 patient's herpesvirus infection symptoms to alleviate; And the every symptom of B group is not all improved.Can obviously find out, after feeding back CTL cell, patients symptomatic is obviously alleviated.
Thus, the CTL cell that known the present invention prepares can promote the overall immunity of patient, obviously improves the symptom of patient, can help that immune response is more complete, usefulness is higher.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. prepare a method for DC cell, it is characterized in that, comprise the following steps:
Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell;
Utilize immature DC cell described in HSV-2 antigen mRNA transfection, to obtain the immature DC cell through transfection; And
The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell,
Wherein, described monocyte is separated to obtain from the peripheral blood mononuclear cell of sample.
2. method according to claim 1, is characterized in that, described HSV-2 antigen mRNA is obtained by following steps:
Preparation HSV-2 antigen cDNA plasmid, the nucleotide sequence of described HSV-2 antigen cDNA is as shown in SEQ ID NO:1;
Described HSV-2 antigen cDNA plasmid is carried out linearization process, to obtain the HSV-2 antigen cDNA plasmid through linearization process; And
The described HSV-2 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain HSV-2 antigen mRNA.
3. method according to claim 2, is characterized in that, with pcDNA3.1 carrier for skeleton carrier, prepares described HSV-2 antigen cDNA plasmid,
Optionally, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site,
Optionally, carry out described linearization process based on restriction enzyme site Bst1107I,
Optionally, T7Message Machine test kit is utilized to carry out described in-vitro transcription,
Optionally, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.
4. method according to claim 1, is characterized in that, utilization electricity turns or PEI method carries out described transfection,
Optionally, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described HSV-2 antigen mRNA,
Optionally, DC substratum is utilized to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation,
Optionally, described DC substratum comprises:
Serum-free cell culture medium;
The CTL4 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once.
5. prepare a method for targeted immune cell mass, it is characterized in that, comprise the following steps:
The peripheral blood mononuclear cell of sample is provided;
The peripheral blood mononuclear cell of described sample is separated into lymphocyte and monocyte;
Described lymphocyte is carried out activation culture, to obtain the lymphocyte through activation culture;
Monocyte is carried out the first differentiation-inducing cultivation, to obtain immature DC cell;
Utilize immature DC cell described in HSV-2 antigen mRNA transfection, to obtain the immature DC cell through transfection;
The described immature DC cell through transfection is carried out the second differentiation-inducing cultivation, to obtain ripe DC cell; And
The DC cell of described maturation and the described lymphocyte through activation culture are carried out mixed culture, to obtain targeted immune cell mass.
6. method according to claim 5, is characterized in that, described HSV-2 antigen mRNA is obtained by following steps:
Preparation HSV-2 antigen cDNA plasmid, the nucleotide sequence of described HSV-2 antigen cDNA is as shown in SEQ ID NO:1;
Described HSV-2 antigen cDNA plasmid is carried out linearization process, to obtain the HSV-2 antigen cDNA plasmid through linearization process; And
The described HSV-2 antigen cDNA plasmid through linearization process is carried out in-vitro transcription, to obtain HSV-2 antigen mRNA,
Optionally, with pcDNA3.1 carrier for skeleton carrier, prepare described HSV-2 antigen cDNA plasmid,
Optionally, described pcDNA3.1 carrier has BamhI, XbaI and Bst1107I restriction enzyme site,
Optionally, carry out described linearization process based on restriction enzyme site Bst1107I,
Optionally, T7Message Machine test kit is utilized to carry out described in-vitro transcription,
Optionally, after carrying out described in-vitro transcription, comprise the step of edulcoration purification further.
7. method according to claim 5, is characterized in that, utilization electricity turns or PEI method carries out described transfection,
Optionally, utilize PEI method to carry out described transfection, wherein, PEI is 3:1 with the mixing quality ratio of described HSV-2 antigen mRNA,
Optionally, described activation culture comprises:
CTL1 cell culture medium is utilized to carry out the first activation culture 24 hours to described lymphocyte;
CTL2 cell culture medium is utilized to carry out the second activation culture 2-3 days to the lymphocyte through the first activation culture; And
CTL3 cell culture medium is utilized to carry out the 3rd activation culture 4-5 days to the lymphocyte through the second activation culture, every equivalent fluid infusion in 2-3 days once,
Optionally, described CTL1 cell culture medium comprises:
Serum-free cell culture medium;
The CTL1 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Described CTL2 cell culture medium comprises:
Serum-free cell culture medium;
The CTL2 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Described CTL3 cell culture medium comprises:
Serum-free cell culture medium;
The CTL3 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, the microbiotic of 0.5 volume % ~ 5 volume % is added with in described serum free medium, preferred gentamicin.
8. method according to claim 5, is characterized in that, utilizes DC substratum to carry out described first differentiation-inducing cultivation and described second differentiation-inducing cultivation,
Optionally, described DC substratum comprises:
Serum-free cell culture medium;
The CTL4 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %, wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, carry out described first differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, carry out described second differentiation-inducing cultivation 4-5 days, every 2-3 days and partly change liquid once,
Optionally, CTL3 cell culture medium is utilized to carry out described mixed culture 6-8 days, preferably 7 days,
Optionally, described CTL3 cell culture medium comprises:
Serum-free cell culture medium;
The CTL3 factor of 100 ~ 1000U/ml; And
The autoserum of 2 volume % ~ 10 volume %,
Wherein, described serum origin in described sample,
Optionally, described serum free medium is the serum free medium of Takara,
Optionally, be added with the microbiotic of 0.5 volume % ~ 5 volume % in described serum free medium, preferred gentamicin,
Optionally, be that 1:20 carries out described mixed culture according to the DC cell of described maturation and the described lymphocytic volume ratio through activation culture.
9. a targeted immune cell mass, it is prepared by the method described in any one of claim 5-8.
10. targeted immune cell mass according to claim 9, and the DC cell utilizing the method preparing DC cell described in any one of claim 1-4 to prepare is preparing the purposes in medicine, described medicine is used for the treatment of HSV-2 to be infected, or suppresses the transfer of HSV-2 positive tumor, diffusion, recurrence
Optionally, described HSV-2 infects for nerpes vinrus hominis infects.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510225001.0A CN104830781A (en) | 2015-05-05 | 2015-05-05 | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510225001.0A CN104830781A (en) | 2015-05-05 | 2015-05-05 | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104830781A true CN104830781A (en) | 2015-08-12 |
Family
ID=53808983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510225001.0A Pending CN104830781A (en) | 2015-05-05 | 2015-05-05 | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104830781A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456945A (en) * | 2018-11-12 | 2019-03-12 | 河北省健海生物芯片技术有限责任公司 | A kind of application for improving DC cell HPV viruse antigen and offering DC cell after the gene modification method and gene modification of efficiency |
| CN109701008A (en) * | 2019-02-18 | 2019-05-03 | 山东兴瑞生物科技有限公司 | For the therapeutic DC combination vaccine and preparation method thereof of herpes simplex virus |
| CN110724181A (en) * | 2019-11-18 | 2020-01-24 | 维塔恩(广州)医药有限公司 | Herpes simplex virus related antigen short peptide and application thereof |
| CN110746497A (en) * | 2019-11-18 | 2020-02-04 | 维塔恩(广州)医药有限公司 | Chlamydia pneumoniae-associated antigen short peptide and application thereof |
| CN110804088A (en) * | 2019-11-18 | 2020-02-18 | 维塔恩(广州)医药有限公司 | Cytomegalovirus-associated antigen short peptide and application thereof |
| CN110845583A (en) * | 2019-11-18 | 2020-02-28 | 维塔恩(广州)医药有限公司 | Varicella zoster virus associated antigen short peptide and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001044477A1 (en) * | 1999-12-17 | 2001-06-21 | American Cyanamid Company | Method of enhancing immune responses to herpes simplex virus vaccine |
-
2015
- 2015-05-05 CN CN201510225001.0A patent/CN104830781A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001044477A1 (en) * | 1999-12-17 | 2001-06-21 | American Cyanamid Company | Method of enhancing immune responses to herpes simplex virus vaccine |
Non-Patent Citations (3)
| Title |
|---|
| COURTNEY THORNBURG 等: "Induction of Cytotoxic T Lymphocytes With Dendritic Cells Transfected With Human Papillomavirus E6 and E7 RNA: Implications for Cervical Cancer Immunotherapy", 《JOURNAL OF IMMUNOTHERAPY》 * |
| TERHUNE,S.S. ET AL: "ACCESSION：U12181.1", 《GENBANK》 * |
| 张素欣: "不同方式负载抗原后外周血树突状细胞对口腔癌抑瘤作用的体内外实验研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456945A (en) * | 2018-11-12 | 2019-03-12 | 河北省健海生物芯片技术有限责任公司 | A kind of application for improving DC cell HPV viruse antigen and offering DC cell after the gene modification method and gene modification of efficiency |
| CN109701008A (en) * | 2019-02-18 | 2019-05-03 | 山东兴瑞生物科技有限公司 | For the therapeutic DC combination vaccine and preparation method thereof of herpes simplex virus |
| CN109701008B (en) * | 2019-02-18 | 2022-06-21 | 山东兴瑞生物科技有限公司 | Therapeutic DC composite vaccine aiming at herpes simplex virus and preparation method thereof |
| CN110724181A (en) * | 2019-11-18 | 2020-01-24 | 维塔恩(广州)医药有限公司 | Herpes simplex virus related antigen short peptide and application thereof |
| CN110746497A (en) * | 2019-11-18 | 2020-02-04 | 维塔恩(广州)医药有限公司 | Chlamydia pneumoniae-associated antigen short peptide and application thereof |
| CN110804088A (en) * | 2019-11-18 | 2020-02-18 | 维塔恩(广州)医药有限公司 | Cytomegalovirus-associated antigen short peptide and application thereof |
| CN110845583A (en) * | 2019-11-18 | 2020-02-28 | 维塔恩(广州)医药有限公司 | Varicella zoster virus associated antigen short peptide and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104830781A (en) | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof | |
| CN101918544B (en) | Method for increasing immunoreactivity | |
| CN102268453B (en) | LMP-1 recombinant adeno-associated virus (rAAV) vector and construction method as well as application thereof | |
| CN104830779A (en) | CEA antigen based DC cell and targeting immune cell population, and preparation method and application thereof | |
| CN104830797A (en) | DC cell based on SP17 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830782A (en) | CK19 antigen based DC cell and targeting immune cell population, and preparation method and application thereof | |
| CN105219711A (en) | The culture system of a kind of CIKs cell and DC-CIKs cell | |
| CN106957822A (en) | Cultural method, kit and the application of amplification in vitro gene editing activating T cell | |
| CN104830792A (en) | DC cell based on BCG1 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830796A (en) | DC cell based on SPANXA1 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830783A (en) | DC cell based on HBV1 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830799A (en) | DC cell based on PSA antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830803A (en) | DC cell based on P53 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN102660579B (en) | HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine | |
| CN104830793A (en) | DC cell based on LMP-1 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830785A (en) | DC cell based on HPVE6 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830791A (en) | DC cell based on PAP antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830794A (en) | DC cell based on HPVE7 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830780A (en) | DC cell based on BA46 antigen and targeting immune cell population, and preparation method and application thereof | |
| CN104830800A (en) | DC cell based on PSMA antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830802A (en) | DC cell based on CA125 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830798A (en) | DC cell based on SCC antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830790A (en) | DC cell based on HM1.24 antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830788A (en) | DC cell based on HBV-HCV antigen, targeting immune cell population, preparation method and applications thereof | |
| CN104830787A (en) | DC cell based on AFP antigen, targeting immune cell population, preparation method and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160729 Address after: 201203 Shanghai City, Pudong New Area Chinese Jing (Shanghai) Free Trade Zone No. 351 Building No. 2 room A656-27 Applicant after: Shanghai Telebio Biomedical Co., Ltd. Address before: 200120 Shanghai Pudong New Area South Road 399, Lane six, room 63, building No. 1501 Applicant before: Yang Guanghua |
|
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150812 |
|
| RJ01 | Rejection of invention patent application after publication |