CN102719400B - Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) - Google Patents
Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) Download PDFInfo
- Publication number
- CN102719400B CN102719400B CN201210231576.XA CN201210231576A CN102719400B CN 102719400 B CN102719400 B CN 102719400B CN 201210231576 A CN201210231576 A CN 201210231576A CN 102719400 B CN102719400 B CN 102719400B
- Authority
- CN
- China
- Prior art keywords
- ctl
- cell
- target
- hla
- rhil
- 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.)
- Active
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 35
- 102000036639 antigens Human genes 0.000 title claims abstract description 33
- 108091007433 antigens Proteins 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 230000000890 antigenic effect Effects 0.000 title claims abstract description 16
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 title claims abstract description 14
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 title description 169
- 210000004027 cell Anatomy 0.000 claims abstract description 122
- 238000000034 method Methods 0.000 claims abstract description 69
- 230000003321 amplification Effects 0.000 claims abstract description 61
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 61
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 43
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 42
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 41
- 239000007790 solid phase Substances 0.000 claims abstract description 11
- 230000004913 activation Effects 0.000 claims abstract description 9
- 230000002494 anti-cea effect Effects 0.000 claims abstract description 9
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 6
- 210000002966 serum Anatomy 0.000 claims description 22
- 230000006698 induction Effects 0.000 claims description 21
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 19
- 239000011324 bead Substances 0.000 claims description 18
- 239000002243 precursor Substances 0.000 claims description 16
- 230000005251 gamma ray Effects 0.000 claims description 15
- 230000009849 deactivation Effects 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 230000010261 cell growth Effects 0.000 claims description 9
- 238000000746 purification Methods 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 230000000638 stimulation Effects 0.000 claims description 7
- 102000004127 Cytokines Human genes 0.000 claims description 5
- 108090000695 Cytokines Proteins 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 238000000684 flow cytometry Methods 0.000 claims description 5
- 230000012010 growth Effects 0.000 claims description 4
- 230000001464 adherent effect Effects 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 238000005192 partition Methods 0.000 claims description 2
- 230000002062 proliferating effect Effects 0.000 claims description 2
- 230000003252 repetitive effect Effects 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 32
- 108010002350 Interleukin-2 Proteins 0.000 abstract description 29
- 229920001184 polypeptide Polymers 0.000 abstract description 17
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 12
- 230000002147 killing effect Effects 0.000 abstract description 9
- 238000009169 immunotherapy Methods 0.000 abstract description 8
- 230000004936 stimulating effect Effects 0.000 abstract 2
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 abstract 1
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 230000005855 radiation Effects 0.000 abstract 1
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 52
- 102000000588 Interleukin-2 Human genes 0.000 description 28
- 210000000612 antigen-presenting cell Anatomy 0.000 description 22
- 102000006354 HLA-DR Antigens Human genes 0.000 description 20
- 108010058597 HLA-DR Antigens Proteins 0.000 description 20
- 102100022297 Integrin alpha-X Human genes 0.000 description 20
- 230000000694 effects Effects 0.000 description 18
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 17
- 102100033467 L-selectin Human genes 0.000 description 17
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 13
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 13
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 102000003812 Interleukin-15 Human genes 0.000 description 10
- 108090000172 Interleukin-15 Proteins 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 8
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 210000000822 natural killer cell Anatomy 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 8
- 102100035793 CD83 antigen Human genes 0.000 description 7
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 239000002504 physiological saline solution Substances 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 6
- 101150013553 CD40 gene Proteins 0.000 description 6
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 6
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 6
- 108010002586 Interleukin-7 Proteins 0.000 description 6
- 102000000704 Interleukin-7 Human genes 0.000 description 6
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 6
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- -1 CD86 Proteins 0.000 description 5
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 5
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 5
- 206010070834 Sensitisation Diseases 0.000 description 5
- 230000006044 T cell activation Effects 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 230000003203 everyday effect Effects 0.000 description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 5
- 230000008313 sensitization Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000013589 supplement Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 4
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 230000005859 cell recognition Effects 0.000 description 4
- 230000000139 costimulatory effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 231100000225 lethality Toxicity 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 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
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 230000030741 antigen processing and presentation Effects 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 210000003071 memory t lymphocyte Anatomy 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 2
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 2
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 102000006992 Interferon-alpha Human genes 0.000 description 2
- 108010047761 Interferon-alpha Proteins 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- 102000013462 Interleukin-12 Human genes 0.000 description 2
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 230000017488 activation-induced cell death of T cell Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000007503 antigenic stimulation Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229940117681 interleukin-12 Drugs 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000015654 memory Effects 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 230000004719 natural immunity Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000005070 ripening Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 229960005486 vaccine Drugs 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
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 206010027457 Metastases to liver Diseases 0.000 description 1
- 101100235006 Mus musculus Lctl gene Proteins 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 208000007660 Residual Neoplasm Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
Images
Abstract
The invention discloses a preparation method of an HLA-A0201 restrictive anti-CEA antigenic specificity CTL. The preparation method includes collecting peripheral blood mononuclear cells (PBMCs) through hemapheresis, and enriching and purifying CD8+T lymphocytes; stimulating the CD8+T lymphocytes by utilizing mature dendritic cells loaded with the HLA-A0201 restrictive CEA antigen polypeptides and promoting T cells to grow by using rhIL-2 and rhIL-7; purifying a target CTL by utilizing a tetramer marking method and a fluorescene-activated cell sorting; stimulating the target CTL to grow with an anti-human CD3 monoclonal antibody coated by solid phase and IL-2; adding autologous PBMCs after radiation of gamma rays to strengthen activation of the target CTL; and adding rhIL-15 for breeding and amplification, collecting and identifying. The prepared target CTL has high purity, multiplication capacity, killing activity and proportion of CTL-CM, and can be applied to immunotherapy such as tumors.
Description
Technical field
The invention belongs to biotechnology exploitation and Applied research fields, relate to the restricted anti-CEA Peptide-specific CTL preparation method of HLA-A0201.
Background technology
One, the predicament of oncotherapy and chance
Malignant tumour has become mankind's number one " killer ", and operation, radiotherapy and chemotherapy are three large ordinary methods for the treatment of tumour, and effective ameliorate tumor is loaded in a short time, but still can not effectively remove tumour cell.MRD, to the part sensitivity of radiotherapy, chemotherapy and resistance, be the root of tumor recurrence and transfer, and recurrence with shift tumour patient main causes of death just.Conventional treatments is unable to do what one wishes, and oncotherapy technology and the product of development of new are extremely urgent.
Two, the birth of cellular immunization treatment technology and development
Play developing rapidly of immunology and oncology the eighties in last century, research group headed by nineteen eighty-two America NI H Rosenberg professor develops killer cell (LAK) immunotherapy of lymphokineactivation, and in follow-up study, the clinical application of LAK cell has been carried out to deep discussion.But LAK Execution is strong not, clinical application needs a large amount of infusions (3 * 10
10-11), increase on the other hand limited in one's ability, need to heavy dose ofly in infusion cell apply interleukin II (IL-2) (100,000 IU/kg, q8h), thereby produce relevant untoward reaction.Then Rosenberg has proposed again tumor infiltrating lymphocyte (TIL), and it kills knurl ability and is significantly improved compared with LAK, and without heavy dose of IL-2 combined utilization, but cell need to be from tumor tissues separated acquisition, this greatly limits its clinical application widely.On above working foundation, the employing interferon-gammas such as Schmidt-Wolf (IFN-γ) of Stanford university in 1991, IL-2 and mouse-anti people CD3 monoclonal antibody (Mouse anti-HumanCD3mAb) have induced the cell mass with powerful anti-tumor activity jointly, called after cytokine induced kill cell (CIK).
Dendritic cell (DCs) is the known up to now interior the most powerful full-time antigen presenting cell (APC) of function of body, there is abundant antigen capture molecule in its surface, antigen presentation molecule (MHC I class and MHC class Ⅱmolecule), co-stimulators (CD80, CD83, CD86, CD40 etc.).DC cell after antigenic stimulation moves to lymphoglandula, and the antigenic information of being carried passes to corresponding T lymphocyte, starts, excites CD4+/CD8+T cellullar immunologic response, specifically killing off tumor cells.Simultaneously the DCs after antigenic stimulation can secreting leukocytes mesonium-8(IL-8), interferon alpha (IFN-α), interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α) etc., enhancing body nonspecific immune response (natural immunity), therefore DC has the laudatory title of " natural immunity adjuvant ", its discoverer Ralph professor Steinman obtains Nobel Prize in medicine in 2011.In view of DC residing Central Position in immunity of organism system of defense, the anti-tumor vaccine based on DC exploitation has made it to become one of most advanced, most promising immunotherapy of tumors technology, and recent domestic scholar has carried out exploring widely to it.2 professors of Stanford medical college in 1992 have set up first hand in the world take DC as basic tumor vaccine company (Dendreon, CA), and the product P rovenge of the said firm has obtained FDA approval listing on April 29th, 2010.But DC function is in vivo subject to the immunological status of patient own, the impact of tumor microenvironment, and in tumour patient body, there are a large amount of supressors, so in the body of DC, effect is also not so good as in vitro effects ideal.
It is the immunotherapy technology of rising in the recent period that cytotoxic T lymphocyte (CTL) builds, and because it has ability special, direct killing tumour target cell, therefore becomes the focus of research.
Three, the effect of carcinomebryonic antigen (CEA) in immunotherapy of tumors
Carcinomebryonic antigen (Carcinoembryonic antigen, CEA) is that nineteen sixty-five is found in intestinal canal tumour and embryo's digestive tube first, 686 amino acid, consists of, and molecular weight is 18～20kD.Recently studies confirm that CEA is one of member of ig supergene family, may participate in cell recognition and react with iuntercellular, and CEA or a kind of adhesion factor, can promote tumour cell and Normocellular combination, and the transfer of tumour is played an important role.Therefore CEA is not only a kind of simple tumor markers, and is the molecule relevant with malignancy of tumorization, in kinds of tumors, expresses.Surpass 90% colorectal carcinoma expression CEA, especially, in Patients with Liver Metastasis, change of serum C EA concentration raises in 80% Patients with Liver Metastasis.The positive rate of Serum Obtained From Advance Gastric Cancer CEA is in 40% left and right, and with by stages relevant, with course advancement, raises gradually.The patients with lung cancer of 40%-80% occurs that CEA raises, and the reactivity of CEA level and treatment and prognosis have good dependency.CEA also can produce from mammary tissue.Therefore CEA can be used as effective target antigen of immunotherapy of tumors.
The M & M of the lung cancer of CEA antigen high expression level, cancer of the stomach, colorectal cancer, mammary cancer is all tumours of rank front three, and does not also have the medicine listing for CEA antigen at present, so development of new is imperative for the medicine of CEA target spot.The exploitation of anti-CEA specific CTL is a kind of developing direction.
Four, the CTL mechanism of action
1, body t cell immune response process
There is huge T cell bank in body, by the different outside antigen molecule (bacterium, virus and tumor-antigen peptide) of the different φt cell receptor of its surface expression (TCR) specific recognition, be activated as thering is the CTL of kill target cell, remove infection and the tumour cell of bacterium, virus, and can produce immunological memory CTL, prevent the invasion again of bacterium, virus and the recurrence of tumour.
CTL immunne response is roughly divided four-stage:
(1) antigen recognition: APC identifies and capture antigen (bacterium, virus, tumour cell) by surface receptor;
(2) antigen processing and submission: antigen through APC digestion, be cracked into peptide molecule, the latter abundant MHC-I class or class Ⅱmolecule in APC born of the same parents are combined and are formed respectively MHC-I-polypeptide or MHC-II-polypeptide complex, and are transported to APC surface;
(3) t cell activation (dual signal theory): APC and T cell meet, T cell is by the MHC-I/II-polypeptide complex of self TCR identification APC submission, CD8+T cell recognition MHC-I-polypeptide complex wherein, CD4+T cell recognition MHC-II-polypeptide complex, the stimulus signal (first signal) that T cell has been accepted antigen adds that immune costimulatory signal (second signal) starts activation, and having cytotoxic effect is CTL;
(4) target cell is killed: activation CTL is circulated to position, antigen place, after the TCR specific recognition antigen presentation cell by CTL surface, kills and wounds and removes.
But, found to have a large amount of immunosuppressive factors in tumour patient body, affect effective activation and the propagation of CTL, quantity is very few, is not enough to contend with the tumour of rapid propagation.If can prepare in vitro a large amount of Peptide-specific CTLs and feed back to patient, can be directly, quick killing tumor cell plays therapeutic action, the residual tumor cell that cannot remove conventional treatment is killed, and can prevent recurrence and the transfer of tumour.
2, CTL phenotype and function difference
According to the expression kind of CTL surface molecular, can be divided into amphitypy: central memory-type CTL(CTL
-CM) and effect memory-type CTL(CTL
-EM).
The researchs such as Klebanoff are found: the CTL that phenotype analytical is CD3+CD45RO+CD62L+CCR7+
-CMthere is stronger anti-tumor capacity; Johnson etc. have compared the CTL that MART-1 tcr gene is modified
-CMand CTL
-EM, found that the former the disappear ability of malignant melanoma cell is 10 times of the latter.The results of study such as Berger also show CTL
-CMthe CTL that is CD3+CD45RO+CD62L-CCR7-compared with phenotype after infusion
-EMcan survive the longer time in vivo.
3, CTL technological development present situation
Comprehensively the external technology of preparing of domestic, external CTL can classify as three kinds of methods and source:
(1) tumor infiltrating lymphocyte (TIL) amplification in vitro method
Separated TIL from tumour patient tumor tissues, adds interleukin II (IL-2) to cultivate, the T cell clone of clone's dilution method screening tumour antigen positive, then add mouse-anti people CD3 monoclonal antibody and IL-2 to carry out amplification cultivation acquisition.
Li etc. select the IV phase malignant melanoma patient of the HLA-A201 positive, its tumor tissues is prepared into after single cell suspension, add IL-2 to cultivate, the positive T cell clone of CD8+T+MART-1+ in screening amplifying cells after 5 weeks, add again the PBMC feeder cell amplification cultivation after mouse-anti people CD3 monoclonal antibody and irradiation, add IL-2 next day, then cultivate and can obtain CTLs in 12 days, can and kill and wound the T2 cell strain specific recognition of load MART-1 polypeptide.
(2) tcr gene is modified method
TCR is the key molecule that T cell recognition antigen, mediated immunity are replied, comprise α, β, γ, tetra-chains of δ, by disulfide linkage, connect into α/β and two kinds of heterodimers of gamma/delta, wherein α/β+T cell accounts for the 90-95% of periphery blood T cell, is main immune effector cell.Tcr gene is modified and is passed through the autologous T lymphocyte of exogenous plasmid transfection, imports the tcr gene of energy identification specificity antigenic peptide, selects positive colony, and amplification cultivation obtains CTL in vitro.Comparatively conventional plasmid has retroviral plasmid or slow virus plasmid.
Morgan etc. adopt anti-CD3+CD28+ magnetic bead to activate CD8+T cell, and application slow virus plasmid transfection is modified MART-1 or the specific tcr gene of gp100, adopts mouse-anti people's CD3 monoclonal antibody and IL-2 culture system to prepare CTLs, 12 days energy amplification cultivation to 10
9individual cell, for the treatment of the melanoma patients of 15 routine HLA-A2+, result shows 2 routine patients and has occurred obvious clinical efficacy, and 1 example 52 years old male patient's armpit metastatic lesion disappears, and hepatic metastases focus has dwindled 89%, and the disease free survival phase is 21 months; 1 example 30 years old male patient, lung's metastatic lesion disappears, and the disease free survival phase is 20 months.
Perro etc. adopt Interleukin-15 (IL-15) and IL-21 (IL-21) combination of cytokines to promote the T cell of the non-propagation of tcr gene transfection, can maintain T cell high expression level CD62L and CD28.
(3) exo-antigen sensitization method
With artificial APC(Artificial APC), or the external repetitious stimulation T cell of antigen (protein, polypeptide, or full cell), Peptide-specific CTL obtained.
The patent of invention of the Zhejiang University (patent No.: CN102168066A) the method for external evoked hepatitis b virus specificity cell toxicity T lymphocyte adopts the method.The coated magnetic bead of MHC-I-Ig and anti-CD28mAb is built to artificial APC, after epitope peptide load APC, external sensitization 3-4 week, then with magnetic bead fractionation by adsorption Peptide-specific CTL, the Tetramer evaluation of dyeing.
The defect of above-mentioned three kinds of methods:
(1) TIL amplification in vitro method
1. need the fresh tumor tissues that obtains patient to originate as CTL, only limit to the patient that can perform the operation, and tumor tissues source is limited;
2. TIL is separated, and obtaining with authentication method of ctl clone is complicated, and in tumor tissues, various cellular constituents are more, more complicated, and the time of isolated cell is long, generally all needs more than 1 month;
3. in TIL, be mixed with CD4+CD25+Treg subgroup, it can suppress CTL amplification efficiency;
4. because in-vitro separation proliferation time is long, increased the chance of polluting.
(2) tcr gene is modified method
1. the introducing of exogenous plasmid (retrovirus or slow virus etc.), brings certain risk to clinical application;
2. endogenous TCR disturbs, and the tcr gene of importing may be able to not import the site of expection, and with endogenic TCR, wrong row occurs, and it can identify antigen is not desired design, and is unknown, has very large risk.
(3) external artificial APC Antigen sensitization method
1. introduce allogenic material: artificial APC, whether the CTL of preparation has that artificial APC is residual is still query;
2. the cycle of the artificial external sensitization CTL of APC longer, need 3-4 week, obtain the needs that CTL also needs could meet after amplification cultivation clinical treatment, so the cycle of vitro culture is long, the probability of pollution is larger.
At present also do not have reported in literature to cross the preparation method of the restricted Peptide-specific CTL of HLA-A201 that preparation time is short, amplification times is high, CTL purity is high and fragmentation effect is good.
Summary of the invention
The object of the invention is to provide for above-mentioned technical problem that a kind of preparation time is short, amplification times is high, CTL purity is high and the preparation method of the restricted anti-CEA Peptide-specific CTL of HLA-A201 that fragmentation effect is good.
The object of the invention is to realize by following technical proposal:
The preparation method of the restricted Peptide-specific CTL of HLA-A0201, the method comprises the following steps:
A. enrichment and the purification process of the first step CTL precursor cell derived cell:
With automatic mononuclearcell Acquisition Instrument (Dan Caiyi), gather peripheral blood mononuclear cells (PBMC) as cell derived; Adopt the negative magnetic bead partition method of clinical grade, enrichment from PBMC, purifying CTL precursor cell are CD8+T lymphocyte.
B. target CTL induction and first round amplification:
With the autologous mature dendritic cell (mDC) of the restricted CEA antigenic peptide of load HLA-A0201, stimulate CD8+T lymphocyte, not only offer the first signal (antigen) of T cell activation, DC offers the necessary second signal of T cell activation (co-stimulators CD40, CD80, CD83 etc.) simultaneously; Add two kinds of cytokines of rhIL-2 and rhIL-7 to combine simultaneously and promote T Growth of Cells, suppress the apoptosis reaction (AICD) of activation induction, extend the life cycle of active cells.
C. target CTL purification process:
In the first round, with the mDC of Antigen, stimulate and increase after target CTL quantity, employing Tetramer marking method and Flow cytometry again purification of target CTL(target CTL refer to the CD8+T that expresses identification CEA antigenic peptide TCR, be Tetramer+CD8+T lymphocyte), select Tetramer+CD8+T, reject the T cell of non-specific amplification, to reduce its impact on target CTL propagation.
D. second of target CTL take turns amplification:
Adopt the anti-human-CD3mAb of solid-phase coating and growth and the propagation of IL-2 stimulation target CTL; Add again the autologous PBMC through gamma-ray irradiation to strengthen the activation to target CTL; Add rhIL-15 can promote the propagation of memory t cell, on the other hand apoptosis capable of inhibiting cell on the one hand.
E. second take turns the rear target CTL phenotypic evaluation of amplification:
Adopt flow cytometry to carry out phenotype analytical to the target CTL having prepared, according to the molecular spectra difference of its expression by Memory CTL (CTL centered by its minute
-CM: CD8+CD45RO+CD62L+CCR7+) with responsiveness CTL(CTL
-EM: CD8+CD45RO+CD62L-CCR7-).
Described preparation method, the target antigen in step b is the preferred 9-15 of a CEA antigenic peptide length amino acid, further the HLA-A0201 restricted CEA antigenic peptide of preferred sequence as shown in SEQ ID NO.1.
Described preparation method, the preparing by following method from body maturation DC of load target antigen in step b: add the mixing normal people AB serum of rhGM-CSF, rhIFN α, deactivation and RPMI1640 culture medium culturing after PBMC is adherent three days, the DC of results adds target antigen to hatch and get final product again.
Described preparation method, the autologous PBMC of the gamma-ray irradiation increasing for target CTL in step c prepares by following method: leave and take the PBMC that part list is adopted acquisition, after the gamma-ray irradiation of 30-50GY, preserve; Preserve liquid: containing 20%(V/V) DMSO and 20%(V/V) the RPMI1640 nutrient solution of normal people AB serum, storage temperature :-80 ℃ of very low temperature are preserved.
Described preparation method, the CD3 monoclonal antibody clonal antibody (anti-human-CD3mAb increasing for target CTL in steps d, be called for short CD3 monoclonal antibody) be following operation: by CD3 monoclonal antibody solid-phase coating in the vessel surface for amplifying cells, CD3 monoclonal antibody can be combined with the CD3 molecule on a plurality of CTL surface, promote the formation of CTL cell clone, promote cells contacting, enter proliferating cycle fast.
Described preparation method, the consumption of each step moderate stimulation molecule or cell is respectively:
A. the target CTL first round each add-on that increases: rhIL-2 final concentration is 500-750IU/ml, and rhIL-7 final concentration is 50-75ng/ml, DC with initial T cell quantity than being 1:5.
B. target CTL second takes turns amplification: rhIL-2 final concentration is 200-500IU/ml, and rhIL-15 final concentration is 100-250ng/ml, and the coated concentration of CD3 monoclonal antibody is 1.0-2.5 μ g/ml, and the PBMC of gamma-ray irradiation is 2:1 with initial CTL quantity ratio.
Described preparation method, after each step operation, cell quantity and phenotypic characteristic are in Table 1.
Table 1
Below the more detailed explanation to preparation method's of the present invention key step:
1, CTL precursor cell purifying and enriching method:
Conventionally adopting periphery PBMC is the lymphocytic source of CD8+T as CTL precursor cell, in PBMC, contain the various kinds of cell compositions such as CD4 positive t lymphocytes (CD4+T), CD8 positive t lymphocytes (CD8+T), bone-marrow-derived lymphocyte, monocyte (Mo), natural killer cell (NK), wherein CD8+T starting quantity directly affects the ultimate capacity of target CTL.
Adopt that the method for singly adopting is disposable gathers a large amount of periphery PBMC, obtain a large amount of initiator cells, unnecessary PBMC can irradiation or frozen, and for repeatedly CTL preparation and application, and method is easy and simple to handle.
Adopt the negative back-and-forth method of clinical grade magnetic bead purifying CD8+T from PBMC, reject CD4+T, B, NK cell, reduce the interference of its rapid amplifying to CD8+T amplification growth.By the negative back-and-forth method of magnetic bead, the purity of CD8+T can be improved to 2-4 doubly (being increased to average more than 90%), see accompanying drawing 1.
2, target CTL induction and first round amplification:
Patent of the present invention adopts three days ripe DC preparation methods, and prepared by DC simple and direct, the time is short, and its form, phenotype and conventional 7-10 days DC comparison no significant differences, are shown in accompanying drawing 2.
Patent of the present invention with load target antigen from body maturation DC, give the CD8+T first signal of activation; DC is rich in co-stimulators simultaneously, gives the CD8+T second signal of activation, guarantees that CD8+T is induced to differentiate into target CTL efficiently under dual signal effect.Associating rhIL-2 and rhIL-7 increase and are better than the effect of monofactor (IL-2) CD8+T, and IL-7 promotes memory t cell clone's propagation, suppress the AICD reaction because causing after long-time IL-2 activating T cell, extend the T cells survival cycle.
3, target CTL purifying and enriching method:
After first round induction amplification, the CD8+T that in CD8+T cell, target CTL expresses identification target antigen (being the restricted CEA antigenic peptide of HLA-A0201) TCR only accounts for part ratio, and ratio is not high, be the CD8+T of non-specific amplification in a large number, without the effect of identification, removing target cell.The present invention adopts that Tetramer marking method is separated with Flow cytometry, purification of target CTL, and enrichment can be identified the CTL of target antigen again, and these CTL just possess the ability of killing and wounding target cell.
After purifying, the purity of target CTL reaches more than 95%.And the several method of conventional report adopts limiting dilution assay mostly, the methods such as electroinjection, time cost is longer, complicated operation, specificity is relatively low.See accompanying drawing 3.
4, target CTL second takes turns amplification:
To T cell amplification, adopt the mouse-anti people CD3/CD28 monoclonal antibody of liquid phase and IL-2 combination to increase more.The present invention adopts solid phase mouse-anti people CD3 monoclonal antibody (anti-human-CD3mAb) coating technique, combines through the autologous PBMC of gamma-ray irradiation and rhIL-15 as the multiple signal that stimulates T cell proliferation.Solid phase CD3-mAb can promote T cell clonal formation, accelerates Growth of Cells; IL-15 makes CTL have anti-apoptosis, promotes the ability of memory T cell growth; The a large amount of costimulatory moleculeses of PBMC surface expression are also secreted cytokine profiles the good growing environment of CTL are provided, and after gamma-ray irradiation, can not breed and disturb the growth of CTL, thereby guarantee the efficient amplification of CTL.
With conventional amplification system comparison: target CTL output prepared by this patent method is high, CTL
cMratio is high, kills and wounds target cell ability stronger.See accompanying drawing 4-5.
Beneficial effect of the present invention:
The present invention combines efficient, simple and direct singly the adopting with magnetic bead sorting method of employing and obtains than the CD8+T of ordinary method a greater number as target CTL precursor cell; By the dendritic cell of the restricted CEA antigenic peptide of HLA-A0201 load, as sensitinogen, associating rhIL-2 and rhIL-7 induction CTL precursor clone are to target CTL differentiation and first round amplification; Through the tetramer (tetramer) mark and fluidic cell sorting technology, from the thin group of mixed C D8+T lymph of sensitization, isolate target CTL again, by adopting solid phase anti-human-CD3-mAb, the PBMC of gamma-ray irradiation is as feeder cell, and associating IL-2 and IL-15 carry out second to target CTL and take turns efficient amplification.The target CTL that adopts the inventive method to prepare possesses high purity, high proliferation ability, and High Fragmentation is active, at high proportion CTL
-CMfeature, can be used for the immunotherapy of CEA associated malignancies and communicable disease.
The present invention adopts different methods to pass through the secondarily purified and enrichment to the restricted anti-CEA Peptide-specific CTL of HLA-A0201, by different amplification systems, the target CTL of enrichment is carried out to two and take turns the induction of amplification and function, make target CTL in purity, quantitatively with in function be all better than several CTL preparation method of the prior art, be embodied in especially the following aspects:
1, the invention provides natural, efficient activated T cell dual signal
1. antigen-specific signal: DCs is the generally acknowledged interior the strongest full-time antigen presenting cell of function of body, body series imitates, copy antigen is caught the process of submission in vivo by DC, select dominant antigen peptide load live body DC as sensitinogen, provide the MHC-polypeptide complex approaching most in human immunity answering space structure, for T cell activation provides specificity first signal.
2. costimulatory signal: the DC costimulatory molecules of expressed in abundance simultaneously, for T cell activation provides natural second signal.
2, the invention provides efficient, simple and direct CTL precursor and target CTL enrichment and purification process
1. the enrichment of CTL precursor cell: it is CD8+T lymphocyte that employing is singly adopted with the negative magnetic bead sorting CTL of clinical grade precursor cell, easy and simple to handle, starting quantity is sufficient, and the CD8+T cell purity of acquisition reaches more than 90%, for preparing q.s, enough purity target CTL provide basic guarantee.
2. the purifying of target CTL cell: adopting Tetramer mark and airflow classification technology, is that Tetramer+CD8+T carries out separation and purification to target CTL, simple and direct, efficient, save time (4-5 hour), purity is up to more than 95%.
3, the invention provides two take turns, efficient target CTL amplification system, to guarantee to provide sufficient amount target CTL
1. first round induction and amplification: adopt the DC of polypeptide load, combine two kinds of cytokine IL-2 and IL-7 amplification system, amplification times reaches 30-50 doubly in two weeks.
2. second take turns target CTL amplification: adopt solid phase mouse-anti people CD3 monoclonal antibody, associational cells factor IL-15 and autologous PBMC amplification system, within 10 days, left and right amplification times is average 40 times, is up to 60 times.Can obtain 10
9individual target CTL cell, reaches the needs of clinical application, and ordinary method (background technology is mentioned three kinds of external evoked CTL that reach same quantity of method needs 3-4 week.
4, the external vigor that kills and wounds of target CTL that prepared by the present invention is higher
The restricted anti-CEA Peptide-specific CTL of HLA-A0201 prepared by the present invention is external can specific recognition and kill and wound the target cell of expressing corresponding antigens, and the high day ordinary method of kill rate, and effect target kill rate when identical on average exceeds 15% left and right.See accompanying drawing 5.
M1 is ordinary method one: the target CTL first round increase and sorting purifying with this patent method, target CTL second takes turns when amplification, with mix the RPMI1640 perfect medium re-suspended cell of normal people AB serum containing 10% deactivation, cell density is 2 * 10
6/ ml, is seeded to 75cm
2in Tissue Culture Flask, every bottle of 30ml, adding final concentration is the mouse-anti people CD3 monoclonal antibody (liquid phase) of 50ng/ml and the rhIL-2 that final concentration is 500IU/ml, mixes gently and is placed in 37 ℃, in 5%CO2 cell culture incubator, cultivates.Every day observation of cell growth conditions, supplement fresh culture in good time and containing 10% deactivation, mix the RPMI1640 perfect medium of normal people AB serum, 200IU/ml rhIL-2, cell proliferation is to a certain amount of expansions bottle.Be cultured to the 10th day harvested cell.With physiological saline washing 2 times and resuspended, calculate the cell quantity obtaining.Get 3 * 10
5individual left and right cell, adds anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody to carry out labeling CT L, detects the ratio of analyzing CD8+CD45RO+CD62L+CCR7+T with flow cytometer.And the kill capability to target cell.Average acquisition CTL cell total amount is (1.0 ± 0.3) * 10
9, CTL
cMratio average out to: 31.9 ± 6.5%, CTL lethality average out to 23.7 ± 4.3% when effect target ratio is 30:1.
M2 is ordinary method two: the target CTL first round increase and sorting purifying with this patent method, target CTL second takes turns when amplification, with mix the RPMI1640 perfect medium re-suspended cell of normal people AB serum containing 10% deactivation, cell density is 2 * 10
6/ ml, is seeded to 75cm
2in Tissue Culture Flask, every bottle of 30ml, adds 1.5 * 10
7the rhIL-2 that the anti-CD3/28 magnetic bead of/ml and final concentration are 500IU/ml, mixes gently and is placed in 37 ℃, in 5%CO2 cell culture incubator, cultivates.Every day observation of cell growth conditions, supplement fresh culture in good time and containing 10% deactivation, mix the RPMI1640 perfect medium of normal people AB serum, 200IU/ml rhIL-2, cell proliferation is to a certain amount of expansions bottle.Be cultured to the 10th day harvested cell.With physiological saline washing 2 times and resuspended, calculate the cell quantity obtaining.Get 3 * 10
5individual left and right cell, adds anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody to carry out labeling CT L, detects the ratio of analyzing CD8+CD45RO+CD62L+CCR7+T and the kill capability to target cell with flow cytometer.Average acquisition CTL cell total amount is (1.2 ± 0.3) * 10
9, CTL
cMratio average out to: 33.4 ± 3.2%, CTL lethality average out to 30.2 ± 4.1% when effect target ratio is 30:1.
M3 is this patent method, on average obtains CTL cell total amount for (3.7 ± 0.3) * 10
9, CTL
cMratio average out to: 75.3 ± 4.9%, CTL lethality average out to 41.3 ± 3.3% when effect target ratio is 30:1.
5, CTL in the target CTL cell that prepared by the present invention
cMratio is high
In CTLs prepared by the present invention, express the centre type memory CTL ratio of CD3+CD45RO+CD62L+CCR7+ phenotype higher than ordinary method, see accompanying drawing 4.
Amplification ability, phenotype and the lethality of target CTL prepared by three kinds of amplification systems of comparison, result shows: the CTL amplification ability of this patent induction system induction is the strongest, and cell total amount can reach (3.7 ± 0.3) * 10
9, CTL wherein
cMratio can reach 75.3%, at effect target, than when the 30:1, for the kill capability of target cell (the T2 cell strain of load HLA-A2-CEA polypeptide), on average can reach 41.3%.
Accompanying drawing explanation
Fig. 1: the comparison of purity before and after CTL precursor cell purifying.
Figure A: singly adopt PBMC fluidic cell and detect collection of illustrative plates (before purifying)
Method: get respectively 50 μ l PBMC (approximately 3 * 10
5individual cell) add in 3 streaming pipes, in the first pipe, add again the anti-human CD4 antibody of 5 μ l FITC marks, the anti-human CD8 antibody of 5 μ l PE marks, the anti-human CD56 antibody of the anti-CD3antibody of 5 μ l PerCP marks and 5 μ lAPC marks; The anti-human CD14 antibody that adds 5 μ l APC marks in the second pipe; The 3rd pipe adds the anti human CD 19 antibody of 5 μ l APC marks, fully mix, 4 ℃ of lucifuges are hatched 30 minutes, after taking out every pipe add 1ml precooling containing 2%(V/V) twice of the physiological saline washing of new-born calf serum and resuspended after, with FACS Calibur flow cytometer, detect, adopt Cellquest software analysis CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3-CD56+% ratio.
Result:
CD14+%（Mo）：9.99% CD19+%（B）：6.15%
CD3+CD4+T%：10.91% CD3+CD8+T%：45.01%
CD3-CD56+%（NK）：0.45%
After negative selection of figure B magnetic bead, CTL precursor cell fluidic cell detects collection of illustrative plates (after purifying)
Method: singly adopt PBMC and select with CD4+CD19+CD16/56+T sorting magnetic bead is negative, reject after CD4+T cell, NK cell and B cell, collect not the streaming dyeing process before according to purifying in conjunction with magnetic bead cell and dye, analyze the variation of CD3+CD4+T%, CD3+CD8+T%, CD14+%, CD19+%, CD3+CD56+%, CD3-CD56+% ratio.Result demonstration, after the negative selection of magnetic bead, CD3+CD8+T% ratio reaches 90.97%, and purity has improved two times.
Result:
CD3+CD4+T%：2.65% CD3+CD8+T%：97.13%
CD14+%（Mo）：0.04% CD19+%（B）：1.01%
CD3-CD56+%（NK）：0.00%
Fig. 2: the DC phenotype comparison of two kinds of different methods inductions
Method: get respectively the 50 μ l rhGM-CSF+rhIFN-α inductions DC(approximately 3 * 10 of 3 days
5individual cell) add in 4 streaming pipes, the first pipe adds the CD40 of 5 μ lPE marks, the anti-human CD11c antibody of the HLA-DR of 5 μ l PerCP marks and 5 μ l APC marks, the second pipe adds the anti-human CD80 antibody of 5 μ l PE marks, the anti-human CD11c antibody of the anti-human HLA-DR antibody of 5 μ l PerCP marks and 5 μ lAPC marks, the 3rd pipe adds the anti-human CD83 antibody of 5 μ l PE marks, the anti-human CD11c antibody of the anti-human HLA-DR antibody of 5 μ l PerCP marks and 5 μ l APC marks, the 4th pipe adds the anti-human CD86 antibody of 5 μ l PE marks, the anti-human CD11c antibody of the anti-human HLA-DR antibody of 5 μ l PerCP marks and 5 μ l APC marks, fully mix, 4 ℃ of lucifuges are hatched 30 minutes, after taking out every pipe add 1ml precooling containing 2%(V/V) twice of the physiological saline washing of new-born calf serum and resuspended after, with FACS Calibur flow cytometer, detect, adopt Cellquest software analysis HLA-DR+CD11c+%, CD40+HLA-DR+CD11c+%, CD80+HLA-DR+CD11c+%, CD83+HLA-DR+CD11c+%, CD86+HLA-DR+CD11c+% ratio.The rhGM-CSF+rhIL-4 induction DC of 7 days detects with same method.Result demonstration, the rhGM-CSF+rhIFN-α induction DC purity phenotype (HLA-DR+CD11c+) of 3 days that this patent adopts is 90.52%, ripening degree phenotype (CD83+) is 77.23%.The rhGM-CSF+rhIL-4 induction DC purity phenotype of 7 days is 90.95%, and ripening degree phenotype is 77.29%, both no significant differences.
Result:
3 days DC phenotype flow cytometer detection collection of illustrative plates of figure A:rhGM-CSF+rhIFN α induction:
HLA-DR+CD11c+%：90.52% CD40+HLA-DR+CD11c+%：87.15%
CD80+HLA-DR+CD11c+%：82.27% CD83+HLA-DR+CD11c+%：77.23%
CD86+HLA-DR+CD11c+%：87.47%
7 days DC phenotype flow cytometer detection collection of illustrative plates of figure B:rhGM-CSF+rhIL-4 induction:
HLA-DR+CD11c+%：90.95% CD40+HLA-DR+CD11c+%：86.85%
CD80+HLA-DR+CD11c+%：88.76% CD83+HLA-DR+CD11c+%：77.29%
CD86+HLA-DR+CD11c+%：88.21%
Fig. 3: the target CTL first round increase after flow cytometer detection collection of illustrative plates
Figure A: the target CTL first round afterwards cell flow cytometer detection collection of illustrative plates (before purifying) that increase
Figure B: the target CTL first round afterwards cell flow cytometer detection collection of illustrative plates (after purifying) that increase
Method: the CTL first round increases and with the Tetramer of PE mark HLA-A2+CEA+ and the anti-human CD8 antibody of FITC mark, the CTL of amplification carried out to mark afterwards, adopt selected by flow cytometry apoptosis Tetramer+CD8+CTL cell, before sorting, Tetramer+CD8+T purity is 3.13%, reaches 95.14% after sorting.
The comparison of CTL phenotype prepared by tri-kinds of different methods of Fig. 4
Figure A(M1): CTL phenotype flow cytometer detection collection of illustrative plates prepared by liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system
Figure B(M2): liquid phase CD3/28 magnetic bead, CTL phenotype flow cytometer detection collection of illustrative plates prepared by IL-2 amplification system
Figure C(M3): solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15, CTL phenotype flow cytometer detection collection of illustrative plates prepared by autologous PBMC amplification system
Method: the target CTL after purifying is divided into 3 parts, first part adds final concentration 50ng/ml mouse-anti people's CD3 monoclonal antibody and 500IU/ml IL-2 to carry out amplification cultivation, second part adds final concentration 10 μ g/ml CD3/28 magnetic beads and 250IU/mlIL-2 to carry out amplification cultivation, the 3rd part adds the pre-coated culturing bottle spending the night of mouse-anti people CD3 monoclonal antibody 2 μ g/ml, according to the ratio of target CTL:PBMC=1:2, add the PBMC through gamma-ray irradiation, and add the IL-2 of final concentration 500IU/ml and final concentration 25ng/ml IL-15 to carry out amplification cultivation.Be cultured to the 7th day results CTL.Get 50 μ lCTL (approximately 3 * 10
5individual cell) add in 2 fluid-guiding type pipes, the first pipe adds the anti-human CD62L antibody of 5 μ l FITC marks, the anti-human CCR7 antibody of 5 μ lPE marks, the anti-human CD45RO antibody of 5 μ lAPC marks, the second pipe adds the anti-CD3antibody of 5 μ lPerCP marks and the anti-human CD8 antibody of 5 μ lPE marks, fully mix, 4 ℃ of lucifuges are hatched 30 minutes, after taking out every pipe add 1ml precooling containing 2%(V/V) twice of the physiological saline washing of new-born calf serum and resuspended after, with FACS Calibur flow cytometer, detect, adopt Cellquest software analysis CD3+CD8+T%, CD45RO+CD62L+% and CCR7+CD62L+% ratio, obtain the phenotype streaming figure of CTL, the CD3+CD8+T% of CTL prepared by this patent method is 97.24%, wherein CD45RO+CD62L+% reaches 74.76%, CCR7+CD62L+% reaches 77.52%, apparently higher than method 1 and 2.
The comparison of CTL kill capability prepared by Fig. 5 different methods is (with CEA
691-699cTL is example).
CTL kill capability prepared by figure A (M1) liquid phase mouse-anti people CD3 monoclonal antibody+IL-2 amplification system
Figure B (M2) liquid phase CD3/28 magnetic bead, CTL kill capability prepared by IL-2 amplification system
Figure C (M3) solid phase mouse-anti people CD3 monoclonal antibody, IL-2, IL-7, IL-15, CTL kill capability prepared by autologous PBMC amplification system
Method: target cell 1: be load C EA
691-699the HLA-A2+T2 cell strain of polypeptide, target cell 2: be the unloaded cell strain of HLA-A2+T2, target cell 3:K562 (strain of NK sensitive cells).
The CTL of embodiment 1 preparation mixes than the ratio of 3:1,10:1 and 30:1 according to effect target with above-mentioned 3 kinds of target cells respectively, and mtt assay detects CTL killing activity.Result demonstration, CTL prepared by the inventive method when effect target ratio is 10:1, reaches 20.5%, apparently higher than method 1 and 2 to the specific killing activity of target cell.
Specific implementation method
The invention will be further elaborated by the following examples.
Embodiment 1CEA
691-699the preparation of antigenic peptide specific CTL
1, HLA-A201+CEA+ colorectal cancer patients is prepared
Extract patient's periphery anticoagulation 1ml, add the HLA-A2-mAb of FITC mark, through the abbreviation of flow cytometer detection HLA-A2(HLA-A201, lower same) expression; Detect CEA antigenic expression.HLA-A2 and CEA express positive simultaneously and are selected in.
2, the restricted CEA antigenic peptide of HLA-A201 is synthetic
Site is 691-699, and sequence is IMIGVLVGV(SEQ ID NO.1) 9 peptides, hereinafter to be referred as CEA
691-699polypeptide, by chemosynthesis (the biochemical company limited of Shanghai gill), fully dissolves with aseptic double-distilled water, and peptide concentration is 5mg/ml, and packing is stored in-80 ℃.
3, PBMC gathers
With singly adopting instrument collection patient periphery PBMCs, through the separated PBMCs of Ficoll density gradient centrifugation, part is for DC induction, and part is for the enrichment of CTL precursor cell and purifying, and part is through gammairradiation frozen in-80 ℃.
4, DC induction and antigen load
Adherent method is prepared DC: PBMCs is suspended in RPMI1640 substratum, and cell density is 3 * 10
6/ ml, inoculating cell is in 75cm
2in culturing bottle, in 5%CO
2, in 37 ℃ of incubators, hatch 90 minutes, with pre-warm saline, wash gently and wash 2-3 time.Attached cell is for DC induction.
DC induction: in culturing bottle, add 30ml DC inducing culture containing 10%(volume ratio, lower with) the mixing normal people AB serum of deactivation, the RPMI1640 perfect medium of final concentration 200ng/ml rhGM-CSF, final concentration 500ng/ml rhIFN-α.Induce and gather in the crops DCs after 3 days.
Polypeptide DC load: the DC of results is suspended in RPMI1640 substratum, and cell density is 1 * 10
6/ ml is also inoculated in cell cultures 6 orifice plates, and every hole 3ml adds CEA in hole
691-699polypeptide to final concentration is 10 μ g/ml, is positioned over 37 ℃, 5%CO
2in incubator, educate altogether results after 2 hours, wash DC bis-times, then use RPMI1640 substratum resuspended with RPMI1640 substratum, cell density is 1 * 10
6/ ml is the restricted anti-CEA Peptide-specific CTL of HLA-A0201 for target CTL() stimulation.
Autologous PBMC for the gamma-ray irradiation of target CTL amplification prepares by following method: leave and take the PBMC that part list is adopted acquisition, through the average 40GY of 30-50GY() gamma-ray irradiation after preserve; Preserve liquid: containing 20%(V/V) DMSO and 20%(V/V) the RPMI1640 nutrient solution of normal people AB serum, storage temperature :-80 ℃ of very low temperature are preserved.
5, the isolation and purification of CTL precursor cell
In the DC results frozen PBMC that recovered the same day, with precooling containing 2% deactivation, mix the PBS solution washing cell 2 times of normal people AB serum, and re-suspended cell, adjusting cell density is 2 * 10
7/ ml, in cell, add clinical grade CD4+CD19+CD16/56+T sorting magnetic bead (Miltenyi CliniMACS), educate altogether mistake post after 30 minutes, collect the CD8+T lymphocyte flowing out, with RPMI 1640 substratum of precooling, wash 2 times, cell is resuspended in containing 10% deactivation and is mixed in normal people AB serum RPMI1640 perfect medium, and cell density is 1 * 10
6/ ml.
6, the target CTL first round increases
The CD8+T of purifying is inoculated in cell cultures 6 orifice plates, and every hole adds 3ml, then adds 0.6ml DC(step 4 preparation of load polypeptide), add IL-2 and IL-7 to make final concentration reach respectively 500IU/ml and 50ng/ml, after mixing gently, put into 5%CO
2, in 37 ℃ of cell culture incubators, cultivate; Every day observation of cell growth conditions, supplement fresh culture (mixing normal people AB serum RPMI1640 perfect medium, 500IU/ml IL-2 and 50ng/ml IL-7 containing 10% deactivation) in good time.The 7th day, the DC(of load polypeptide was the same in every Kong Zhongzai, to add 0.6ml) again stimulate.Every day observation of cell growth conditions, supplement fresh culture (ditto) in good time, when cell density is excessive, expand bottle, cell proliferation can be moved to 75cm after a certain amount of
2in culturing bottle, continuing amplification cultivates.
7, target CTL sorting purifying
The DC of load polypeptide and CD8+T educate 12-14 days altogether, harvested cell, and the PBS solution washing cell that contains 2% deactivation mixing normal people AB serum of use precooling 2 times, and re-suspended cell, adjusting cell density is 1 * 10
7/ ml adds the HLA-A201+CEA of PE mark in cell suspension
691-699the CD8mAb of Tetramer and FITC mark after mixing gently, educates altogether 20 minutes in 4 ℃ of refrigerators.With precooling containing 2% deactivation, mix the PBS solution washing cell 2 times of normal people AB serum, re-suspended cell, adjusting cell density is 1 * 10
7/ ml, carries out airflow classification by cell, and results CD8+Tetramer+T lymphocyte is abandoned supernatant after centrifugal, and with mix the RPMI1640 perfect medium re-suspended cell of normal people AB serum containing 10% deactivation, cell density is 2 * 10
6/ ml.
8, target CTL second takes turns amplification
Tissue Culture Flask is coated: in step 7 the day before yesterday, get 75cm
2tissue Culture Flask, in wherein add 2ml mouse-anti people CD3 monoclonal antibody (concentration 2 μ g/ml, producer: U.S. R & D company, catalog number (Cat.No.): 59-MAB100, clone number: C1UCHT1, lower with), coated the spending the night of the rearmounted 4 ℃ of refrigerators of sealing;
Before inoculating cell, take out coated culturing bottle, unnecessary CD3-mAb in culturing bottle is abandoned in suction, every bottle adds 30ml sorting CD8+Tetramer+T lymphocyte, add through 2 times of the PBMC(of gamma-ray irradiation quantity to target CTL), add final concentration 500IU/ml IL-2 and final concentration 250ng/ml IL-15, mix gently and be placed in 37 ℃, 5%CO
2in cell culture incubator, cultivate.Every day observation of cell growth conditions, supplement fresh culture (mixing normal people AB serum, 200IU/ml IL-2, RPMI 1640 perfect mediums of 100ng/ml IL-15 containing 10% people's deactivation) in good time and control cell density; Cell proliferation is to a certain amount of expansion bottle.
9, target CTL results and Phenotypic examination
Second takes turns target CTL amplification about 10 days, harvested cell.With physiological saline washing 2 times, be resuspended in 100ml physiological saline, cell density is 10
7/ ml, for immunotherapy.
Get 3 * 10
5individual left and right cell, add anti-human CD8, CD45RO, CD62L, CCR7 monoclonal antibody to carry out labeling CT L, with flow cytometer, detect the ratio of analyzing CD8+CD45RO+CD62L+CCR7+T, the results are shown in Figure 4C, the CD3+CD8+T% of CTL prepared by present method is 97.24%, wherein CD45RO+CD62L+% reaches 74.76%, CCR7+CD62L+% and reaches 77.52%.
After each step operation, cell quantity and phenotypic characteristic are in Table 1.
10, target CTL kill capability
By preparation target CTL respectively with upper load C EA
691-699the HLA-A2+T2 cell strain of polypeptide, the unloaded cell strain of HLA-A2+T2 and three kinds of target cells of K562 (strain of NK sensitive cells) mix than the ratio of 3:1,10:1 and 30:1 according to effect target, and mtt assay detects CTL killing activity.Result demonstration, CTL prepared by the inventive method when effect target ratio is 10:1, reaches 20.5% to the specific killing activity of target cell, sees Fig. 5 C.
Claims (5)
1.HLA-A0201 restricted anti-CEA Peptide-specific CTL preparation method, is characterized in that the method comprises the following steps:
Enrichment and the purifying of a.CTL precursor cell derived cell:
The collection peripheral blood mononuclear cells of singly gathering is originated as CTL precursor cell; Adopt the negative magnetic bead partition method of clinical grade, enrichment from collect peripheral blood mononuclear cells, purifying CTL precursor cell are CD8+T lymphocyte;
B. target CTL induction and first round amplification:
With load HLA the mature dendritic cell of the restricted CEA antigenic peptide of A0201 stimulate CD8+T lymphocyte, add simultaneously rhIL 2 and rhIL 7 two kinds of cytokines combine and promote T Growth of Cells; Second week again repetitive stimulation once, completes first round amplification, described HLA the restricted CEA antigenic peptide of A0201 sequence as shown in SEQ ID NO.1; Wherein load HLA the mature dendritic cell of the restricted CEA antigenic peptide of A0201 by following method, prepare: after peripheral blood mononuclear cells is adherent, add containing rhGM the mixing normal people AB serum of CSF, rhIFN α, deactivation and RPMI1640 culture medium culturing three days, results DC adds target antigen to hatch and get final product again.
C. target CTL purification process:
Adopt Tetramer marking method and Flow cytometry purification of target CTL, select Tetramer+CD8+T lymphocyte;
D. target CTL second takes turns amplification:
Adopt solid-phase coating anti human CD3mAb and IL the growth of 2 stimulation target CTL; Add the autologous peripheral blood mononuclear cells after gamma-ray irradiation to strengthen the activation to target CTL; Add rhIL 15 continue to cultivate, complete second and take turns amplification, collect and identify.
2. preparation method according to claim 1, it is characterized in that in step c preparing by following method for the autologous peripheral blood mononuclear cells of the gamma-ray irradiation of target CTL amplification: leave and take part claim 1 and singly adopt acquisition peripheral blood mononuclear cells, through 30 preserve after the gamma-ray irradiation of 50GY; Preserve liquid: containing 20%(V/V) DMSO and 20%(V/V) the RPMI1640 nutrient solution of normal people AB serum, storage temperature: 80 ℃ of very low temperature preserve.
3. preparation method according to claim 1, it is characterized in that in steps d for the anti of target CTL amplification human CD3mAb be following operation: by anti human CD3mAb solid-phase coating in the vessel surface for amplifying cells, anti human CD3mAb be combined with the CD3 molecule on a plurality of CTL surface, promote the formation of CTL cell clone, promote cells contacting, enter proliferating cycle fast.
4. preparation method according to claim 1, is characterized in that the consumption of each step moderate stimulation molecule or cell is respectively:
A. the target CTL first round increase each add-on: rhIL 2 final concentrations be 500 750IU/ml, rhIL 7 final concentrations be 50 75ng/ml, DC with initial CD8+T cell quantity than being 1:5;
B. target CTL second take turns amplification: rhIL 2 final concentrations be 200 500IU/ml, rhIL 15 final concentrations be 100 250ng/ml, anti human the coated concentration of CD3mAb be 1.0 2.5 μ g/ml, the peripheral blood mononuclear cells of gamma-ray irradiation with initial CTL quantity than being 2:1.
5. preparation method according to claim 1, is characterized in that after each step operation that cell quantity and phenotypic characteristic are respectively:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210231576.XA CN102719400B (en) | 2012-07-05 | 2012-07-05 | Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210231576.XA CN102719400B (en) | 2012-07-05 | 2012-07-05 | Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102719400A CN102719400A (en) | 2012-10-10 |
| CN102719400B true CN102719400B (en) | 2014-04-09 |
Family
ID=46945273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210231576.XA Active CN102719400B (en) | 2012-07-05 | 2012-07-05 | Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102719400B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104072599A (en) * | 2014-06-06 | 2014-10-01 | 深圳市阳溪生物科技有限公司 | Polypeptide, dendritic cell adopting in vitro activation and application of dendritic cell |
| CN104830779A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | CEA antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
| CN105368784A (en) * | 2015-08-26 | 2016-03-02 | 上海宇研生物技术有限公司 | Lung cancer CTL cell culture reagent kit and preparation method |
| RU2619186C1 (en) * | 2016-03-31 | 2017-05-12 | Федеральное государственное бюджетное научное учреждение "Научно-исследовательский институт фундаментальной и клинической иммунологии" (НИИФКИ) | Method for production of in vitro populations of activated antigenspecific antitumor-tumor cytotoxic t-lymphocytes specific to tumor-associated antigen epitopes |
| WO2019196087A1 (en) * | 2018-04-13 | 2019-10-17 | Syz Cell Therapy Co. | Methods of cancer treatment using tumor antigen-specific t cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618498A (en) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003225866A1 (en) * | 2002-03-22 | 2003-10-13 | Aventis Pasteur, Inc. | Peptide epitopes recognized by antigen specific cd8+ t lymphocytes |
-
2012
- 2012-07-05 CN CN201210231576.XA patent/CN102719400B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618498A (en) * | 2012-03-26 | 2012-08-01 | 时宏珍 | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) |
Non-Patent Citations (8)
| Title |
|---|
| Cassian Yee et al.Isolation of High Avidity Melanoma-Reactive CTL from Heterogeneous Populations Using Peptide-MHC Tetramers.《Journal of immunology》.1999,第162卷(第4期),2227-2234. |
| Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8+ CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells;Shengming Dai et al;《Jounral of Immunology》;20061231;第84卷(第12期);全文 * |
| IL-15-induced human DC efficiently prime melanomaspecific naive CD8+ T cells to differentiate into CTL;Peter dubsky et al.;《Eur. J. Immunol》;20071231;第37卷(第6期);1678-1690 * |
| Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers;Susann Szmania et al;《Blood》;20010831;第98卷(第3期);505-512 * |
| Isolation of High Avidity Melanoma-Reactive CTL from Heterogeneous Populations Using Peptide-MHC Tetramers;Cassian Yee et al;《Journal of immunology》;19991231;第162卷(第4期);2227-2234 * |
| Peter dubsky et al..IL-15-induced human DC efficiently prime melanomaspecific naive CD8+ T cells to differentiate into CTL.《Eur. J. Immunol》.2007,第37卷(第6期),1678-1690. |
| Shengming Dai et al.Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8+ CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells.《Jounral of Immunology》.2006,第84卷(第12期),1067-1076. |
| Susann Szmania et al.Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers.《Blood》.2001,第98卷(第3期),505-512. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102719400A (en) | 2012-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102618498B (en) | Preparation method for HLA-A0201 limited antigen specificity CTL (cytotoxic T lymphocyte) | |
| AU2006328943B2 (en) | Improved method for expansion of tumour-reactive T-lymphocytes for immunotherapy of patients with cancer | |
| JP6869994B2 (en) | Medium addition kit for NK cell culture, and NK cell culture method using the kit | |
| CN102719402B (en) | Preparation method of HLA-A0201-restricted anti-HPV (human papillomavirus) antigen-specific CTL | |
| CN102719400B (en) | Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) | |
| CN103923880A (en) | Efficient multiplication CTL preparation method killing tumors in targeted mode | |
| CN105524884A (en) | Preparation method of HLA-A0201 restriction AFP antigen specific CTL | |
| CN109161527A (en) | A kind of efficient NK methods for cell expansion | |
| CN104072599A (en) | Polypeptide, dendritic cell adopting in vitro activation and application of dendritic cell | |
| CN102321581A (en) | Preparation method of ascites tumor cell sensitized DC-CIK | |
| CN102719401B (en) | Preparation method of HLA-A0201 (Human Leukocyte Antigen-A0201) restrictive anti-MAGE (MicroArray and Gene Expression) antigenic specificity CTL (Cytotoxic T Lymphocyte) | |
| CN105524883A (en) | Capri cell and preparation method thereof | |
| Aglietta et al. | Ex vivo expansion of hematopoietic cells and their clinical use | |
| CN104894072A (en) | Preparation method and application of autologous natural killer cell proliferation | |
| CN109957543A (en) | Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact | |
| CN105505871B (en) | A kind of effective amplification CIK and improve the method that its specificity kills tumor ability | |
| CN105969731B (en) | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions | |
| CN105505873A (en) | Preparation method of HLA-A0201 restriction anti-Her2-neu antigen-specific CTL (cytotoxic lymphocyte) | |
| CN109535241B (en) | DC-CIK (dendritic cell-cytokine induced killer) co-culture cell, preparation method thereof, sensitizing antigen and application | |
| CN110628717B (en) | Method for culturing infiltrating T cells | |
| CN113293130B (en) | Culture method of tumor specific T cells | |
| CN105219727A (en) | A kind of test kit for activating colorectal cancer specific immune response | |
| CN105624110A (en) | Preparation method for HLA-A0201 restrictive anti-PSA antigen-specificity CTL | |
| CN105219717A (en) | An a kind of type polarization dendritic cell and induction method thereof and application | |
| CN103911341B (en) | The preparation method of Th1 cell subsets and preparing the application in antitumor cell preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20121010 Assignee: Jiangsu Kang Biotechnology Co.,Ltd. Assignor: Shi Hongzhen Contract record no.: 2014320000369 Denomination of invention: Preparation method of HLA-A0201 restrictive anti-carcinoembryonic antigen (CEA) antigenic specificity cytotoxic T lymphocyte (CTL) Granted publication date: 20140409 License type: Exclusive License Record date: 20140430 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| ASS | Succession or assignment of patent right |
Owner name: JIANGSU DECON BIO-SCI-TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SHI HONGZHEN Effective date: 20141230 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 211100 NANJING, JIANGSU PROVINCE TO: 211899 NANJING, JIANGSU PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20141230 Address after: 211899, Jiangsu Province, Pukou District, Nanjing Pu Road, No. 28, building 1, new mile 5 Patentee after: Jiangsu Kang Biotechnology Co.,Ltd. Address before: Jiangning District of Nanjing City, Jiangsu province 211100 General Road No. 10, Tsinghua Yuan 58 Room 201 Patentee before: Shi Hongzhen |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20180925 Address after: 210000 room 208, block A, building 10, Spark Road, Pukou District, Nanjing, Jiangsu. Patentee after: Lu Anjun Address before: 211899 5, building 1, new mileage 28, Tianpu Road, Pukou District, Nanjing, Jiangsu, China Patentee before: Jiangsu Kang Biotechnology Co.,Ltd. Effective date of registration: 20180925 Address after: 210000 B-6, Jiangsu science and Technology Park, 9 Wei Di Road, Qixia District, Nanjing, Jiangsu. Patentee after: De Kang cell bioengineering Center (Jiangsu) Co.,Ltd. Address before: 210000 room 208, block A, building 10, Spark Road, Pukou District, Nanjing, Jiangsu. Patentee before: Lu Anjun |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230110 Address after: Building B6-1, Jiangsu Life Science and Technology Innovation Park, No. 9, Weidi Road, Qixia District, Nanjing, Jiangsu, 210000 Patentee after: Jiangsu Kang Biotechnology Co.,Ltd. Address before: 210000 B-6, Jiangsu science and Technology Park, 9 Wei Di Road, Qixia District, Nanjing, Jiangsu. Patentee before: De Kang cell bioengineering Center (Jiangsu) Co.,Ltd. |
|
| TR01 | Transfer of patent right |