WO2023277086A1 - 血中循環腫瘍細胞を含む試料を調製する方法 - Google Patents
血中循環腫瘍細胞を含む試料を調製する方法 Download PDFInfo
- Publication number
- WO2023277086A1 WO2023277086A1 PCT/JP2022/026036 JP2022026036W WO2023277086A1 WO 2023277086 A1 WO2023277086 A1 WO 2023277086A1 JP 2022026036 W JP2022026036 W JP 2022026036W WO 2023277086 A1 WO2023277086 A1 WO 2023277086A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- circulating tumor
- antibody
- cells
- analysis
- tumor cells
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 177
- 210000004369 blood Anatomy 0.000 title claims abstract description 92
- 239000008280 blood Substances 0.000 title claims abstract description 92
- 210000004881 tumor cell Anatomy 0.000 title abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 160
- 238000004458 analytical method Methods 0.000 claims abstract description 108
- 238000002617 apheresis Methods 0.000 claims abstract description 95
- 210000001616 monocyte Anatomy 0.000 claims abstract description 65
- 230000003321 amplification Effects 0.000 claims abstract description 21
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 21
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 18
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 18
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 18
- 230000035755 proliferation Effects 0.000 claims abstract description 8
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 claims description 149
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 91
- 108020004414 DNA Proteins 0.000 claims description 60
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 48
- 210000004443 dendritic cell Anatomy 0.000 claims description 46
- 230000005574 cross-species transmission Effects 0.000 claims description 36
- 238000003559 RNA-seq method Methods 0.000 claims description 29
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 108010026552 Proteome Proteins 0.000 claims description 18
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 17
- 238000005194 fractionation Methods 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 12
- 238000002659 cell therapy Methods 0.000 claims description 11
- 101000613490 Homo sapiens Paired box protein Pax-3 Proteins 0.000 claims description 10
- 102100040891 Paired box protein Pax-3 Human genes 0.000 claims description 10
- 238000011222 transcriptome analysis Methods 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 230000000869 mutational effect Effects 0.000 claims description 3
- 238000000575 proteomic method Methods 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 abstract description 8
- 239000011886 peripheral blood Substances 0.000 abstract description 8
- 239000002609 medium Substances 0.000 description 46
- 239000000523 sample Substances 0.000 description 46
- 238000005119 centrifugation Methods 0.000 description 45
- 206010028980 Neoplasm Diseases 0.000 description 35
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 33
- 201000011510 cancer Diseases 0.000 description 27
- 239000012503 blood component Substances 0.000 description 25
- 239000006228 supernatant Substances 0.000 description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 239000000427 antigen Substances 0.000 description 20
- 108091007433 antigens Proteins 0.000 description 20
- 102000036639 antigens Human genes 0.000 description 20
- 239000006285 cell suspension Substances 0.000 description 20
- 210000002381 plasma Anatomy 0.000 description 19
- 238000012163 sequencing technique Methods 0.000 description 18
- 239000007788 liquid Substances 0.000 description 17
- 238000000926 separation method Methods 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 16
- 239000000306 component Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 238000002955 isolation Methods 0.000 description 14
- 239000008188 pellet Substances 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 210000004698 lymphocyte Anatomy 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- 238000000684 flow cytometry Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000005138 cryopreservation Methods 0.000 description 9
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 239000012595 freezing medium Substances 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 210000001772 blood platelet Anatomy 0.000 description 8
- 238000013507 mapping Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 206010009944 Colon cancer Diseases 0.000 description 7
- 238000007400 DNA extraction Methods 0.000 description 7
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 7
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 208000029742 colonic neoplasm Diseases 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 210000000265 leukocyte Anatomy 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000002504 physiological saline solution Substances 0.000 description 7
- 238000010079 rubber tapping Methods 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 6
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 230000005484 gravity Effects 0.000 description 6
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 6
- 201000002528 pancreatic cancer Diseases 0.000 description 6
- 208000008443 pancreatic carcinoma Diseases 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 238000010257 thawing Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 5
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000010195 expression analysis Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 201000007270 liver cancer Diseases 0.000 description 5
- 238000011177 media preparation Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 210000005087 mononuclear cell Anatomy 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 4
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 4
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000002619 cancer immunotherapy Methods 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000007667 floating Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 239000012192 staining solution Substances 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 238000004885 tandem mass spectrometry Methods 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 3
- 208000017604 Hodgkin disease Diseases 0.000 description 3
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 3
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 3
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 3
- 206010060862 Prostate cancer Diseases 0.000 description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 3
- 238000011530 RNeasy Mini Kit Methods 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 101000588258 Taenia solium Paramyosin Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 210000003169 central nervous system Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 3
- 229960002986 dinoprostone Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 201000004101 esophageal cancer Diseases 0.000 description 3
- 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 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 101150073223 hisat gene Proteins 0.000 description 3
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 230000002980 postoperative effect Effects 0.000 description 3
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- 108091092584 GDNA Proteins 0.000 description 2
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 2
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 201000009365 Thymic carcinoma Diseases 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004820 blood count Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013020 final formulation Substances 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000005534 hematocrit Methods 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 208000028830 lung neuroendocrine neoplasm Diseases 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 201000005962 mycosis fungoides Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 208000021284 ovarian germ cell tumor Diseases 0.000 description 2
- 201000003913 parathyroid carcinoma Diseases 0.000 description 2
- 208000017954 parathyroid gland carcinoma Diseases 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000002616 plasmapheresis Methods 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 208000002008 AIDS-Related Lymphoma Diseases 0.000 description 1
- 206010069754 Acquired gene mutation Diseases 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 206010073360 Appendix cancer Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 206010004593 Bile duct cancer Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006143 Brain stem glioma Diseases 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 206010007279 Carcinoid tumour of the gastrointestinal tract Diseases 0.000 description 1
- 208000037138 Central nervous system embryonal tumor Diseases 0.000 description 1
- 206010007953 Central nervous system lymphoma Diseases 0.000 description 1
- 235000005956 Cosmos caudatus Nutrition 0.000 description 1
- 208000009798 Craniopharyngioma Diseases 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 230000009946 DNA mutation Effects 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061825 Duodenal neoplasm Diseases 0.000 description 1
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 208000018058 Ewing sarcoma of bone Diseases 0.000 description 1
- 208000017259 Extragonadal germ cell tumor Diseases 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000021309 Germ cell tumor Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 206010021042 Hypopharyngeal cancer Diseases 0.000 description 1
- 206010056305 Hypopharyngeal neoplasm Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010061252 Intraocular melanoma Diseases 0.000 description 1
- 208000009164 Islet Cell Adenoma Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 206010023825 Laryngeal cancer Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 206010025312 Lymphoma AIDS related Diseases 0.000 description 1
- 206010025557 Malignant fibrous histiocytoma of bone Diseases 0.000 description 1
- 206010073059 Malignant neoplasm of unknown primary site Diseases 0.000 description 1
- 208000032271 Malignant tumor of penis Diseases 0.000 description 1
- 208000002030 Merkel cell carcinoma Diseases 0.000 description 1
- 206010063569 Metastatic squamous cell carcinoma Diseases 0.000 description 1
- 208000032818 Microsatellite Instability Diseases 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 206010029266 Neuroendocrine carcinoma of the skin Diseases 0.000 description 1
- 208000033383 Neuroendocrine tumor of pancreas Diseases 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010067517 Pancreatic neuroendocrine tumour Diseases 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- 206010034299 Penile cancer Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 208000009359 Sezary Syndrome Diseases 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 206010054184 Small intestine carcinoma Diseases 0.000 description 1
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 208000031673 T-Cell Cutaneous Lymphoma Diseases 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 208000033781 Thyroid carcinoma Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 208000023915 Ureteral Neoplasms Diseases 0.000 description 1
- 206010046392 Ureteric cancer Diseases 0.000 description 1
- 206010046431 Urethral cancer Diseases 0.000 description 1
- 206010046458 Urethral neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000008385 Urogenital Neoplasms Diseases 0.000 description 1
- 201000005969 Uveal melanoma Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 208000008383 Wilms tumor Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 208000020990 adrenal cortex carcinoma Diseases 0.000 description 1
- 208000007128 adrenocortical carcinoma Diseases 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 208000021780 appendiceal neoplasm Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical compound COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 201000008873 bone osteosarcoma Diseases 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 208000025188 carcinoma of pharynx Diseases 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 201000007455 central nervous system cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 101150082482 ctc gene Proteins 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 208000017763 cutaneous neuroendocrine carcinoma Diseases 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000008716 dendritic activation Effects 0.000 description 1
- 230000004041 dendritic cell maturation Effects 0.000 description 1
- 229940029030 dendritic cell vaccine Drugs 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 201000000312 duodenum cancer Diseases 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 201000008819 extrahepatic bile duct carcinoma Diseases 0.000 description 1
- 201000009311 eye carcinoma Diseases 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 102000054766 genetic haplotypes Human genes 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 201000003115 germ cell cancer Diseases 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 201000006866 hypopharynx cancer Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 201000008893 intraocular retinoblastoma Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 201000002529 islet cell tumor Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 206010023841 laryngeal neoplasm Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000006178 malignant mesothelioma Diseases 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002705 metabolomic analysis Methods 0.000 description 1
- 230000001431 metabolomic effect Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 208000037830 nasal cancer Diseases 0.000 description 1
- 210000001989 nasopharynx Anatomy 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 201000008026 nephroblastoma Diseases 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 201000002120 neuroendocrine carcinoma Diseases 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000000683 nonmetastatic effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 208000020668 oropharyngeal carcinoma Diseases 0.000 description 1
- 201000006958 oropharynx cancer Diseases 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 208000022102 pancreatic neuroendocrine neoplasm Diseases 0.000 description 1
- 208000021010 pancreatic neuroendocrine tumor Diseases 0.000 description 1
- 210000002568 pbsc Anatomy 0.000 description 1
- 238000005453 pelletization Methods 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- -1 picibanil Proteins 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 210000004623 platelet-rich plasma Anatomy 0.000 description 1
- 210000004910 pleural fluid Anatomy 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 208000016800 primary central nervous system lymphoma Diseases 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 201000003804 salivary gland carcinoma Diseases 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000037439 somatic mutation Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 201000003957 thoracic cancer Diseases 0.000 description 1
- 208000008732 thymoma Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 208000013077 thyroid gland carcinoma Diseases 0.000 description 1
- 230000003614 tolerogenic effect Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 208000018417 undifferentiated high grade pleomorphic sarcoma of bone Diseases 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 208000037965 uterine sarcoma Diseases 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 208000013139 vaginal neoplasm Diseases 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2304—Interleukin-4 (IL-4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
- C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Definitions
- the present disclosure relates to a method of preparing a sample containing circulating tumor cells derived from a subject for analysis without proliferation and a method of analyzing circulating tumor cells derived from a subject without proliferation.
- the present disclosure provides a method for preparing a sample for identifying a neoantigen possessed by a subject, a method for identifying a neoantigen possessed by a subject from the sample, and a cell for dendritic cell therapy using the neoantigen possessed by a subject. It relates to a method of manufacturing.
- Neoantigens also called neoantigens or neoantigens
- neoantigens are mutated antigens newly appearing due to genetic mutations that occur in cancer cells. Since neoantigens are different for each individual patient, they are expected to be applied to personalized medicine.
- neoantigens newly generated in cancer tissues are known to induce strong immune responses, and it has been suggested that they are compatible with cancer immunotherapies such as immune checkpoint inhibitors.
- the present inventors have found a method for identifying neoantigens from patient blood samples without using the amount of tumor tissue conventionally required as a sample for identifying neoantigens.
- the present inventors have also discovered a technique for preparing a sample that can be used for analyzes other than neoantigen identification from a patient's blood sample.
- (Item X1) A method of preparing a sample comprising circulating tumor cells from a subject for analysis without nucleic acid amplification and/or culture expansion, comprising: isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; If necessary, a step of confirming the purity of blood circulating tumor cells necessary for the analysis; optionally, subjecting the blood circulating tumor cells to pretreatment for the analysis.
- (Item X2) A method according to the preceding items, wherein the apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item X3) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item X4) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- (Item X5) The method according to any one of the preceding items, wherein the circulating tumor cells are about 1 ⁇ 10 3 or more.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody.
- (Item X9) The method of any one of the preceding items, wherein the analysis comprises whole exome analysis, whole genome analysis, RNA-Seq, single-cell RNA-Seq, proteome analysis, and transcriptome analysis.
- (Item X10) A method according to any one of the preceding items, wherein the sample is for identifying a neoantigen possessed by the subject by performing the analysis.
- (Item X11) The method of any one of the preceding items, further comprising the step of extracting DNA, RNA, or protein from said circulating tumor cells.
- (Item X12) A method according to any one of the preceding items, wherein at least about 100 pg of the DNA or RNA is extracted.
- (Item X13) A method for analyzing circulating tumor cells derived from a subject without amplification by nucleic acid amplification and/or culture, comprising: isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; and performing an analysis using the circulating tumor cells.
- apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item X15) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item X16) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- (Item X17) The method according to any one of the preceding items, wherein the circulating tumor cells are about 1 ⁇ 10 3 or more.
- (Item X18) The method according to any one of the preceding items, wherein the peripheral blood mononuclear cells are about 1 ⁇ 10 4 or more.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody. (Item X20) The method of any one of the preceding items, further comprising freezing the circulating tumor cells.
- (Item X21) The method of any one of the preceding items, wherein the analysis comprises whole exome analysis, whole genome analysis, RNA-Seq, single-cell RNA-Seq, proteome analysis, and transcriptome analysis.
- (Item X22) A method for identifying a neoantigen possessed by the subject by analyzing the circulating tumor cells derived from the subject by the method according to any one of the above items.
- (Item X23) The method of any one of the preceding items, further comprising the step of extracting DNA, RNA, or protein from said circulating tumor cells.
- (Item X24) further creating a DNA/RNA sequence library from said DNA or RNA; and identifying a neoantigen based on the mutational information of said sequence library, wherein said circulating tumor cells are at least about 20% pure.
- (Item X25) The method of any one of the preceding items, wherein said analysis comprises whole exome analysis of said DNA.
- (Item X26) The method of any one of the preceding items, wherein said analysis further comprises proteomic analysis of said circulating tumor cells.
- (Item X27) A method according to any one of the preceding items, wherein at least about 100 pg of the DNA or RNA is extracted.
- a method for producing cells for dendritic cell therapy using a neoantigen possessed by a subject isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; extracting DNA or RNA from said circulating tumor cells and creating a DNA/RNA sequence library; A step of identifying a neoantigen based on the mutation information of the sequence library; and introducing said neoantigen into dendritic cells.
- (Item X29) A method according to any one of the preceding items, wherein the apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item X30) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item X31) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody.
- the method of any one of the preceding items further comprising freezing the circulating tumor cells.
- Item X36 A method according to any one of the preceding items, wherein at least about 100 pg of the DNA or RNA is extracted.
- a method of treating or preventing a subject by analyzing circulating tumor cells without nucleic acid amplification and/or proliferation in culture comprising: isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; performing an analysis using the circulating tumor cells; and treating or preventing the subject based on the results of the analysis.
- the analysis comprises whole exome analysis, whole genome analysis, RNA-Seq, single-cell RNA-Seq, proteome analysis
- the present disclosure also provides: (Item 1) 1. A method of preparing a sample for identifying neoantigens possessed by a subject by sequencing without nucleic acid amplification and/or growth in culture, comprising: isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; optionally, confirming the purity of the circulating tumor cells necessary for identification of the neoantigen; providing said circulating tumor cells as a sample for identifying neoantigens and/or extracting DNA and/or RNA from said circulating tumor cells.
- (Item 2) A method according to the preceding items, wherein the apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item 3) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item 4) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- (Item 5) The method according to any one of the preceding items, wherein the circulating tumor cells are about 1 ⁇ 10 3 or more.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody.
- (Item 9) A method according to any one of the preceding items, wherein at least about 100 pg of the DNA or RNA is extracted.
- (Item A1) 1. A method of preparing a sample for identifying neoantigens possessed by a subject by sequencing without nucleic acid amplification and/or growth in culture, comprising: isolating monocytes from peripheral blood mononuclear cells obtained from the subject using apheresis; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; optionally, confirming the purity of the circulating tumor cells necessary for identification of the neoantigen; providing said circulating tumor cells as a sample for identifying neoantigens and/or extracting DNA and/or RNA from said circulating tumor cells.
- (Item B1) A method of identifying a neoantigen possessed by a subject by sequencing without nucleic acid amplification and/or propagation in culture, comprising: isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; extracting DNA and/or RNA from said circulating tumor cells and creating a DNA/RNA sequence library; and identifying a neoantigen based on the mutational information of said sequence library, wherein said circulating tumor cells are at least about 20% pure.
- (Item B2) A method according to any one of the preceding items, wherein the apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item B3) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item B4) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- (Item B5) The method according to any one of the preceding items, wherein the circulating tumor cells are about 1 ⁇ 10 3 or more.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody.
- (Item B9) A method according to any one of the preceding items, wherein at least about 100 pg of the DNA or RNA is extracted.
- (Item C1) A method for producing cells for dendritic cell therapy using a neoantigen possessed by a subject, isolating monocytes from peripheral blood mononuclear cells collected from the subject using apheresis, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; isolating circulating tumor cells from the peripheral blood mononuclear cells from which monocytes have been isolated; extracting DNA and/or RNA from said circulating tumor cells and creating a DNA/RNA sequence library; A step of identifying a neoantigen based on the mutation information of the sequence library; and introducing said neoantigen into dendritic cells.
- (Item C2) A method according to any one of the preceding items, wherein the apheresis conditions comprise spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- (Item C3) A method according to any one of the preceding items, wherein the apheresis conditions comprise a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- (Item C4) A method according to any one of the preceding items, wherein the step of isolating the circulating tumor cells is performed using a monoclonal antibody or by size fractionation.
- (Item C5) The method according to any one of the preceding items, wherein the circulating tumor cells are about 1 ⁇ 10 3 or more.
- the monoclonal antibody is an anti-EpCAM antibody, an anti-Vimentin antibody, an anti-N-Cadherin antibody, an anti-CD20 antibody, an anti-E-Cadherin antibody, an anti-Desmoglein-3 antibody, an anti-Syndecan-1 antibody, an anti-CD99 antibody, an anti-CD81 antibody, and The method of any one of the preceding items, comprising a PAX3 antibody.
- a patient's neoantigen can be identified non-invasively.
- the method of the present disclosure can be used for cancer immunotherapy using neoantigens, and can be applied to personalized medicine and regenerative medicine.
- FIG. 1 is a schematic diagram showing the procedure for producing neoantigen dendritic cells and the flow of dendritic cell therapy using the same according to an embodiment of the present disclosure.
- FIG. 2 shows the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure.
- FIG. 3 is a schematic diagram showing the procedure for producing neoantigen dendritic cells and the flow of dendritic cell therapy using the same according to another embodiment of the present disclosure.
- FIG. 4 shows the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure. The results of NY lung cancer (stage 4) patients, CTC20-0065 colon cancer (stage 4) patients, and C-0107 ovarian cancer (stage 4) patients are shown.
- FIG. 4 shows the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure. The results of NY lung cancer (stage 4) patients, CTC20-0065 colon cancer (stage 4) patients, and C-0107 ovarian cancer (stage 4) patients.
- FIG. 5 is a diagram showing the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure. Results for 21-0062 breast cancer patient (postoperative), CTC20-014 pancreatic cancer (stage 4) patient, and C-0082 pancreatic cancer (stage 4) patient are shown.
- FIG. 6 is a diagram showing the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure. 21-0059 Patients with papillary duodenal cancer (Stage 4), CTC 21-0052 Patients with lung cancer (Stage 4).
- FIG. 7 shows the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure.
- FIG. 8 is a diagram showing the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure.
- FIG. 9 shows the results of flow cytometric analysis of cells isolated in one embodiment of the present disclosure. The results of C-0099 (esophageal cancer) (postoperative) are shown.
- Neoantigen is also called neoantigen, neoantigen, or tumor-specific mutated antigen, and refers to antigens generated by DNA mutations that alter proteins in tumor cells. Neoantigens can potentially be perceived as non-self by the immune system.
- apheresis generally refers to separating one or more blood components (plasma components, cell components such as platelets, leukocytes, etc.) from the blood of a subject by extracorporeal circulation. Blood collected by extracorporeal circulation is called apheresis blood. As used herein, apheresis involves extracorporeal circulation of a subject's blood through a blood component separator to collect peripheral blood mononuclear cells.
- the term "buffy coat” refers to the white layer between red blood cells and plasma after whole blood is sedimented by centrifugation.
- Whole blood is separated by centrifugation into plasma (platelet-poor plasma), which is a light specific gravity component (supernatant component), red blood cells (concentrated red blood cells), which is a heavy specific gravity component (sedimentation component), and buffy coat, which is an intermediate specific gravity component.
- plasma platelet-poor plasma
- red blood cells concentrated red blood cells
- buffy coat which is an intermediate specific gravity component.
- the buffy coat contains a leukocyte component and platelet-rich plasma (platelet-containing component).
- the term "buffy coat volume” refers to the amount of buffy coat collected.
- spillover refers to switching between blood return and collection in order to collect the buffy coat when the buffy coat is sufficiently concentrated in apheresis. Spillover can remove excess platelets and lymphocytes. Also, “spillover volume” refers to the amount of buffy coat that is delivered to the harvesting stage.
- blood circulating tumor cell enrichment conditions when referring to apheresis conditions, refer to specific conditions under which apheresis is performed under which circulating tumor cells are enriched in the resulting compartment. say.
- blood circulating tumor cell concentration conditions include spillover volume, buffy coat volume, or a combination thereof, and these values can be achieved by setting these values to specific conditions.
- the spillover volume may be from about 5 to about 15 ml, preferably from about 7 to 14 ml, more preferably from about 9 to about 12 ml.
- the buffy coat volume can be about 5 to about 15 ml, preferably about 6 to about 12 ml, more preferably about 7 to about 10 ml, to achieve circulating tumor cell enrichment conditions.
- peripheral blood mononuclear cells also called PBMCs, refer to any peripheral blood cell with a round nucleus.
- Normal peripheral blood mononuclear cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes. In the present disclosure, it has been found for the first time that peripheral blood mononuclear cell components substantially contain CTCs.
- monocyte is a type of leukocyte and the largest type of leukocyte. Refers to cells that can differentiate into macrophages and dendritic cells. Monocytes are both part of the innate immunity of vertebrates and have an impact on adaptive immune processes. Human blood is said to contain at least three types of monocytes.
- blood circulating tumor cells refers to tumor cells that have penetrated into blood vessels or lymph vessels from primary tumors (primary tumors) or metastatic tumors (metastasis).
- primary tumors primary tumors
- metastatic tumors metastatic tumors
- circulating tumor cells that circulate in the CTCs exist, for example, as tumor cells circulating in the peripheral bloodstream of cancer patients.
- CTCs are blood-based markers thought to have predictive and prognostic functions in cancer detection and progression. Many types of cancer are known to give rise to CTCs.
- CTCs are nucleated, CD45-negative, and cytokeratin-positive cells, and express at least one of tumor markers, sarcoma markers, and osteosarcoma markers. Isolation of CTCs from whole blood is technically challenging because CTCs are so scarce.
- dendritic cells also called DCs, refer to potent antigen-presenting cells for initiation and control of cellular immune responses in humans.
- Dendritic cells can be either immunostimulatory or immunosuppressive, depending on which set of their potential properties they express upon interaction with a responsive-specific clone of T cells. Therefore, it is believed to play a very important central role in T cell-mediated immune responses.
- immature dendritic cells are considered to be more 'tolerogenic' than mature dendritic cells, while mature dendritic cells are considered to be more 'immunogenic' than their precursors.
- Dendritic cells are generated ex vivo from monocytes and CD34 positive cells (myeloid cells).
- tumor or “cancer” is not particularly limited, but for example, breast cancer, gastrointestinal/gastrointestinal cancer, anal cancer, appendix cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor (gist), islet cell tumor, adult primary liver cancer, pediatric liver cancer, pancreatic cancer, rectal cancer, small bowel cancer and gastric cancer; endocrine and neuroendocrine cancers such as pancreatic glands cancer, adrenocortical carcinoma, pancreatic neuroendocrine tumor, Merkel cell carcinoma, non-small cell lung neuroendocrine tumor, small cell lung neuroendocrine tumor, parathyroid carcinoma, pheochromocytoma, pituitary tumor and thyroid carcinoma; ocular carcinoma, e.g.
- Intraocular melanoma and retinoblastoma Intraocular melanoma and retinoblastoma; urogenital cancers such as bladder cancer, kidney (renal cell) cancer, penile cancer, prostate cancer, transitional cell renal pelvic and ureteral cancer, testicular cancer, urethral cancer and Wilms tumor; germ cell cancer , e.g.
- pediatric central nervous system cancer pediatric extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor; Ovarian germ cell tumors, uterine sarcoma, vaginal and vulvar cancers; head and neck cancers such as hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous cell carcinoma of the neck of unknown primary, oral cancer, nasopharynx Cavity carcinoma, oropharyngeal carcinoma, sinus and nasal carcinoma, parathyroid carcinoma, pharyngeal carcinoma, salivary gland carcinoma and throat carcinoma; Acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy cell leukemia; multiple myeloma, including malignant plasma cells; lymphomas, including AIDS-related lymphoma, cutaneous t-cell lymphoma, adult Hodgkin's lymphoma, pediatric Hodgkin's lymphoma, Hodgkin'
- non-small cell lung cancer small cell lung carcinoma, malignant mesothelioma, thymoma and thymic carcinoma; and skin cancers such as Kaposi's sarcoma, melanoma and squamous cell carcinoma, carcinoma of unknown primary.
- identify refers to visualizing the presence of neoantigen.
- to isolate or “isolate” refers to the physical separation of cells of interest such as CTCs from other cell types present in a sample of interest.
- whole exome analysis refers to a method of comprehensively analyzing only exon sequences in the whole genome, and is also simply referred to as "exome analysis”. Specifically, exon regions are enriched from the genome, base sequences are determined using a next-generation sequencer (NGS), etc., and after mapping the obtained lead sequences to reference sequences, SNVs are detected and annotated. Exon sequences can be analyzed by The term “exome analysis” includes the case of analyzing only some exomes by target sequencing that examines only a specific region of DNA.
- whole genome analysis is a technique for analyzing the base sequence of all DNA that makes up the genome of a living organism, and mutations are detected from the obtained base sequence information. Specifically, the extracted DNA is fragmented to prepare a library, and the base sequence is determined (sequenced) using a next-generation sequencer. Mutations are then detected by bioinformatic analysis.
- the term “genome” refers not only to a narrowly defined genome (a set of chromosomes containing the minimum set of genes necessary for living organisms to carry out normal life activities), but also to genomes that do not constitute chromosomes. Those that code genetic information are also included.
- RNA-Seq refers to RNA sequencing or RNA sequencing, which is a technique for decoding the sequences of mRNA and miRNA in cells, quantifying expression levels, discovering novel transcription sequences, and the like. be. This is done by analyzing lead information obtained using a next-generation sequencer.
- Single-cell RNA-Seq is a method of performing RNA sequencing using RNA obtained from a single cell.
- proteome analysis refers to an analysis that elucidates the relationship between genetic information and various proteins that interact in a complex manner in cells, and targets the structure and function of proteins. It is a large-scale analysis method and can comprehensively analyze proteins.
- a proteome is the entire protein produced in a specific cell, organ, or organ. can be detected, it can be used as a method for separating biological samples such as proteins.
- transcriptome analysis refers to all mRNA (or , primary transcripts, transcripts). Since mRNA changes in various ways due to the accumulation of extracellular influences that the cell receives during the process of development, it is possible to analyze the properties of the current cell in detail. Specifically, analysis is performed using a microarray or the like. For example, compared with cells to be compared, circulating tumor cells correspond to mRNAs involved in infinite proliferation, invasion, metastasis, resistance, recurrence, etc. of cancer cells, and mutated oncogenes. When there are many mRNAs, mRNAs corresponding to mutated tumor suppressor genes, and mRNAs for cancer-associated genes, it is possible to identify the properties of circulating tumor cells.
- a method of preparing a sample for identification of neoantigens possessed by a subject by sequencing without nucleic acid amplification and/or expansion in culture comprising peripheral blood mononuclear cells obtained from the subject using apheresis. isolating monocytes from corpuscles, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; and from said peripheral blood mononuclear cells from which monocytes are isolated; a step of isolating circulating tumor cells; optionally confirming the purity of the circulating tumor cells necessary for identifying neoantigens; providing as a sample and/or extracting DNA and/or RNA from said circulating tumor cells.
- a method of preparing a sample for identifying neoantigens possessed by a subject by sequencing without nucleic acid amplification and/or propagation in culture comprising: A step of isolating monocytes from peripheral blood mononuclear cells collected from a subject, a step of isolating blood circulating tumor cells from the peripheral blood mononuclear cells from which the monocytes were isolated, and optionally accordingly, confirming the purity of the circulating tumor cells necessary for identifying the neoantigen and providing the circulating tumor cells as a sample for identifying the neoantigen and/or and extracting DNA and/or RNA from.
- a method of identifying a neoantigen possessed by a subject by sequencing without nucleic acid amplification and/or expansion in culture comprising peripheral blood mononuclear cells obtained from the subject using apheresis. isolating monocytes from corpuscles, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; and from said peripheral blood mononuclear cells from which monocytes are isolated; a step of isolating circulating tumor cells in the blood; extracting DNA and/or RNA from the circulating tumor cells in the blood; creating a DNA/RNA sequence library; and wherein said circulating tumor cells are at least about 20% pure.
- neoantigen identification approximately 30 mg of tumor tissue is usually required by biopsy or during the surgical procedure.
- biopsies and surgical operations on patients are burdensome, and biopsies and surgical operations only for identification of neoantigens are generally not realistic. Therefore, in one embodiment of the present disclosure, less tumor tissue, preferably peripheral blood mononuclear cells obtained from a subject using apheresis, can be used to prepare samples for identifying neoantigens.
- apheresis conditions can include spillover volume and/or buffy coat volume to circulating tumor cell enrichment conditions.
- apheresis conditions can include a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- the apheresis conditions other than the spillover volume and the buffy coat volume may be any settings as long as they are the conditions for enriching circulating tumor cells in the blood, and the default conditions of the device may be used. can be done.
- apheresis can be performed using, for example, a centrifugal blood component separator (Comtech) to separate blood into blood components such as platelets, lymphocytes, leukocytes, plasma, etc., or perform plasmapheresis. be able to.
- a centrifugal blood component separator Comtech
- whole blood is continuously withdrawn from a patient/donor, anticoagulated and defoamed, and separated into major blood components using centrifugal force. The degree of separation depends on the position of the interface, blood flow and centrifugation speed.
- the blood components are separated in the centrifuge according to size and specific gravity in the order of erythrocytes-granulocytes-(monocytes, lymphocytes, stem cells)-platelets from the outside, and plasma is separated in the innermost part.
- the separation chamber has outlets for the various blood components, and the components other than the blood component intended for collection are remixed in the drip chamber and returned to the patient/donor. .
- the chamber is provided with a row of light-passing holes that define the interface boundaries (the outer cell fraction and the inner plasma fraction). ) are automatically detected by an optical sensor.
- the accumulated target blood component undergoes a process of elevating the position of the interface, called spillover, via the plasma exit port, and accumulates in the reservoir bag by the collection pump.
- centrifugal blood component separator (Comtech) The specifications of the centrifugal blood component separator (Comtech) are shown below. 1. Centrifuge inner rotor rotation speed: 300-2200rpm Outer rotor rotation speed: 150-1100rpm Maximum rotation speed: 2200 rpm 2. Pump type: Line roller pump Speed range: 0.3-21 rpm (collection pump, plasma pump, whole blood pump, ACD pump/circulation pump) 1-28 rpm (ACD pump) Flow rate range Blood collection: 12.4-120 mL/min Plasma: 5.12-80 mL/min Collection: 1.78-80 mL/min Anticoagulant: 1.78-17.5 mL/min3.
- Plt-5d collection of platelets for storage up to 5 days by double needle method
- Plt-5d-SN collection of platelets for storage up to 5 days by single needle method
- PBSC Lymphocyte: peripheral blood stem cells and Lymphocyte isolation
- RV-PBSC Minor enriched peripheral blood stem cell collection
- BMSC Stem cell isolation from bone marrow
- Granulocyte Granulocyte collection
- MNC Mononuclear cell collection
- Depletion Plasma or plasma and lymphocyte therapeutic Depletion
- TPE Therapeutic Plasmapheresis Adsorption: Therapeutic Plasma Separation
- RBC Therapeutic Red Blood Cell Exchange and Depletion
- data such as the patient's sex, height, weight, hematocrit value, and white blood cell count can be set.
- the patient's circulating blood volume is determined by gender, height, and weight, and the appropriate blood collection volume is set based on that data. Also, by setting the hematocrit value, the value of the plasma pump can be determined.
- the white blood cell count it is possible to calculate a numerical value for the purpose of how much blood must be processed to form an appropriate buffy coat for efficient collection. In one embodiment, it is also possible to set a numerical value “spillover volume” for efficiently collecting cells during blood collection and a “buffy coat volume” that determines how much blood is to be collected.
- separation of blood components can be performed as follows. (1) Completely degas the saline line and prepare for pre-separation according to the display. (2) Press the (Continue) key to pierce the blood donor with the blood collection needle and collect a pre-donation sample. Puncture the blood return needle. (3) Enter patient information. (4) Press the (Start) key to start separation. Separation and blood collection are performed automatically and automatically terminated when the target setpoint is reached.
- the blood when Comtech is used, the blood can be returned as follows after separating the blood components. (1) Press the (Continue) key to release the clamp of the saline line, disconnect the blood collection line from the donor, and prepare for blood return. (In the case of the single needle method, do not remove the blood collection line.) (2) Press the (Start) key to start blood return. Blood return is performed automatically and ends. (3) After returning blood, press the (Stop) key.
- spillover conditions can be appropriately set. Since the spillover volume greatly affects the collection efficiency of cells, it is adjusted so that cells can be collected efficiently. For example, it can be varied according to the blood condition of the subject, and can be adjusted according to the presence or absence of numbness, the physical condition of the subject, and the like. In one embodiment, the spillover volume can be a value to achieve blood circulating tumor cell enrichment conditions, for example, the spillover volume is about 5 to about 15 ml, preferably about 7 to 14 ml, more preferably about 9 ml. to about 12 ml.
- the buffy coat volume can be appropriately set so that the target cells can be collected.
- the buffy coat volume can be a value to achieve circulating tumor cell enrichment conditions, for example, the buffy coat volume is about 5 to about 15 ml, preferably about 6 to about 12 ml, and further Preferably, it can be about 7 to about 10 ml.
- the spillover volume and the buffy coat volume can be used in combination to achieve blood circulating tumor cell enrichment conditions.
- the centrifugal blood component separation device is not limited to the specific device described above, and may be any device capable of collecting peripheral blood mononuclear cells.
- a centrifugal blood component separator in addition to Comtech, Spectra Optia (Terumo Corporation) and the like can be used.
- the process of separating blood components using Spectra Optia is described below.
- the device is first powered on, the blood circuit is attached to the device, and the circuit is tested and primed. Then follow the instructions on the screen to connect the patient/donor, open the clamps on the blood collection line and the blood return line, and separate the blood components.
- information is displayed on the screen during separation of blood components so that patient/donor status can be monitored at all times, as well as concentration monitoring of the collection port, RBC interface, and collection line.
- concentration monitoring of the collection port, RBC interface, and collection line When the alarm is displayed, or if you want to change the conditions during processing, you can adjust according to the instructions on the screen and restart the operation.
- the component separation process as follows can proceed continuously.
- Whole blood of the patient/donor introduced into the blood collection line attached to the circuit is introduced into the channel portion in the circuit attached to the centrifuge by the action of the blood collection pump.
- An anticoagulant is used at a fixed ratio so that the blood does not clot. Anticoagulated blood is returned to the patient/donor.
- the blood in the channel is separated into components by rotating with the rotation of the centrifuge.
- Each component after centrifugation is separated into three lines of tubes for plasma, platelets, and erythrocytes, flows out of the centrifuge, and enters the cassette section in the circuit by pumping.
- the blood led to the blood return reservoir is returned to the patient/donor through the blood return line by the action of the blood return pump.
- the replacement fluid is introduced into the reservoir in the cassette through the replacement fluid line by the replacement fluid/sampling pump, and supplied to the patient.
- each of the above processes can be monitored by each safety function indicated by a safety device, and can be designed so that each function operates depending on the situation when a situation related to the safety function occurs.
- each operation can be performed on a touch screen type monitor, and patient/donor information and operation programs required for operation can be input to the automatic control computer through this screen.
- the Ethernet port also allows communication with external information equipment, but does not connect electrical equipment to the Ethernet in the patient/donor environment.
- the number of peripheral blood mononuclear cells isolated by apheresis may be sufficient to isolate monocytes or circulating tumor cells, e.g., at least about 10 about 1 ⁇ 10 2 or more, about 1 ⁇ 10 3 or more, about 1 ⁇ 10 4 or more, about 1 ⁇ 10 5 or more, about 1 ⁇ 10 6 or more, about 1 ⁇ 10 7 or more , or about 1 ⁇ 10 8 or more.
- the sample subjected to apheresis can be any sample suitable for identifying neoantigens, including, for example, whole blood, bone marrow, pleural fluid, ascites, central spinal fluid, urine, saliva and bronchial lavage. be able to.
- the sample is blood, such as whole blood or any fraction or component thereof.
- Blood samples suitable for use in the present disclosure may be extracted from any source known to contain blood cells or components thereof, such as arteries, veins, peripheral tissue, umbilical cord, and the like.
- samples for apheresis are obtained and processed using well-known and routine clinical methods (eg, whole blood aspiration and processing procedures).
- an exemplary method can be peripheral blood aspiration from a cancer patient.
- PBMC obtained by apheresis mainly consist of T lymphocytes and monocytes. Isolation of monocytes from PBMCs can be performed using any technique known in the art, for example, in one embodiment of the present disclosure, PBMCs are isolated, seeded, and isolated from circulating tumor cells and lymphocytes. Monocytes can be isolated by collecting the supernatant containing the floating cells present.
- circulating tumor cells can be isolated from peripheral blood mononuclear cells from which monocytes are isolated. Isolation of circulating tumor cells from PBMCs can be performed using any technique known in the art, e.g. CTCs can be isolated by techniques such as isolation with monoclonal antibodies, size fractionation, or gravity fractionation.
- monoclonal antibodies used to isolate CTCs from peripheral blood mononuclear cells from which monocytes have been isolated are, for example, anti-EpCAM antibodies, anti-Vimentin antibodies, anti-N-Cadherin antibodies, anti-CD20 antibodies, anti- E-Cadherin antibodies, anti-Desmoglein-3 antibodies, anti-Syndecan-1 antibodies, anti-CD99 antibodies, anti-CD81 antibodies, and PAX3 antibodies can be included.
- an appropriate antibody can be appropriately selected according to the type of cancer.
- EpCAM-negative pancreatic cancer can be isolated by CD45-Vimentin+ or CD45-N-Cadherin+. .
- isolation of CTCs from peripheral blood mononuclear cells from which monocytes have been isolated can be performed by size fractionation using a cell search system, a metal mesh, or the like.
- a filter kit (ScreeCell) or a metal mesh (Murata Manufacturing) can be used.
- CTC collection filter kit ScreeCell
- CTCs can be collected on the filter during the process of passing whole blood through the filter. Since no antibody is used, EMT (epithelial-mesenchymal transition) cells with little expression of EpCAM (epithelial cell adhesion molecule) can also be collected.
- a metal mesh Morata Seisakusho
- by selecting an appropriate mesh size cells to be captured and cells to be passed can be selected. Only CTCs can be captured by choosing a mesh size suitable for .
- isolation of CTCs from peripheral blood mononuclear cells from which monocytes have been isolated can also be fractionated by specific gravity using an elutriator or the like. Since blood components have different densities, density centrifugation (eg, Ficoll-Paque) (CYTIVA, Marlborough, Mass., USA) can also be used to separate peripheral blood mononuclear cells and CTCs from whole blood. In other embodiments, a system combining a density gradient centrifugation system with a porous barrier that enhances depletion of mononuclear cells, or an immunoaffinity approach to gradient centrifugation to direct unwanted PBMC cells to red blood cells in the sample. By cross-linking and forming an immunorosette, the separation efficiency of CTCs can be greatly improved.
- density centrifugation eg, Ficoll-Paque
- CYTIVA Marlborough, Mass., USA
- CTCs and control cells when isolating CTCs using a monoclonal antibody, CTCs and control cells (antibody-positive cells) can be sorted using a cell sorter.
- a cell sorter SH800 Synchronization
- an example of its operating procedure is as follows.
- sorting can be performed by the following procedure. Thaw frozen cells at 37°C ⁇ Filter Falcon Remove clumped cells ⁇ 3 ml PBS Buffer (2 mM EDTA, 2% FBS) ⁇ Centrifugation 1000 rpm, 2 minutes, 4°C ⁇ Cell count ⁇ Centrifugation 1000 rpm, 2 minutes, 4°C ⁇ Antibody staining Antibody concentration CD45 PE 5 ⁇ g/ml EpCAM 10 ⁇ g/ml ⁇ 30 minutes on ice ⁇ Add 2 ml PBS Buffer (2 mM EDTA, 2% FBS) ⁇ Centrifugation 1000 rpm, 2 minutes, 4°C ⁇ Suspend in 1 ml PBS Buffer (2 mM EDTA, 2% FBS) ⁇ filter, tube ⁇ CML 6ml CML 1% MEM Non-Essential Amino Acid Solution 1% HEPES 1% sodium pyruvate 50 ⁇ M 2-mercaptoethanol 1% Penicillin
- the purity of CTCs isolated by monoclonal antibodies or size fractionation may be sufficient to identify neoantigens or sufficient to extract DNA or RNA. at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, etc. can do.
- the number of CTCs isolated by monoclonal antibodies or size fractionation may be an amount necessary for identifying neoantigens or an amount sufficient for extracting DNA or RNA. It can be at least about 10 or more, about 1 ⁇ 10 2 or more, about 1 ⁇ 10 3 or more, about 1 ⁇ 10 4 or more, or about 1 ⁇ 10 5 or more.
- circulating tumor cells isolated from peripheral blood mononuclear cells can be frozen.
- the blood-circulating tumor cells are cryopreserved at a temperature and time that does not affect the quality that can be provided as a sample for identifying neoantigens or the quality that allows extraction of DNA and/or RNA from the blood-circulating tumor cells.
- it can be cryopreserved in liquid nitrogen ( ⁇ 196° C.) or an ultra-low temperature bath ( ⁇ 80° C.) for about one year or more.
- DNA and/or RNA can be extracted from circulating tumor cells isolated from peripheral blood mononuclear cells.
- the amount of DNA or RNA extracted can be any amount that can be used for DNA/RNA sequencing, eg, at least about 100 pg of DNA and/or RNA can be extracted.
- the extracted amount of DNA and/or RNA may be at least about 200 pg, about 500 pg, about 800 pg, about 1 ng, about 10 ng, about 30 ng, about 50 ng, about 80 ng, about 100 ng, about 200 ng. can.
- DNA extraction can be performed, for example, by the following procedure.
- DNA extraction with QIamp (registered trademark) DNA Mini 20 ⁇ l Proteinase K ⁇ 1.5 ml microtube ⁇ Add the sample suspended in 200 ⁇ l of PBS to this tube ⁇
- 200 ⁇ l Buffer AL Mix well with vortex ⁇ Incubate at 56°C for 10 minutes ⁇
- Add 500 ⁇ l Buffer AW1 ⁇ Close the lid and centrifuge 6,000 g for 1 minute ⁇
- Add 500 ⁇ l Buffer AW2 ⁇ Close the lid and centrifuge 12,000 g for 3 minutes ⁇ Discard the filtrate and centrifuge at 12,000 g for 1 minute
- DNA extraction can be performed, for example, by the following procedure.
- NucleoSpin registered trademark
- Blood L TAKARA
- Proteinase K Add and dissolve Proteinase Buffer PB (stored at -20°C)
- Wash Buffer B5 Add 100% ethanol ⁇
- Use sample maximum 2 ⁇ 10 7 cells
- cells pellet
- BQ1 Vigorously vortex 10 seconds
- Incubate at 56°C for 15 minutes ⁇ Preheat the heat block ⁇
- RNA extraction can be performed using kits and techniques known in the art, for example, the Rneasy mini kit (QIAGEN) can be used.
- the Rneasy mini kit QIAGEN
- RNA can be extracted by the following procedure.
- the following procedure can be employed when using Kiazol and Trizol to extract RNA.
- Thaw frozen cells ⁇ Suspend in 3 ml of PBS (2% FBS, 2 mM EDTA) ⁇ Centrifugation 1000 rpm, 2 minutes, 4°C ⁇ Cell count 1.91 ⁇ 10 6 cells ⁇
- Divide into 2 tubes of 500 ⁇ l each (15 ml tube) ⁇ Centrifugation 1000 rpm, 2 minutes, 4°C ⁇ QIAzol Lysis reagent 700 ⁇ l TRIzol Reagent 700 ⁇ l Suspend in each ⁇ 140 ⁇ l of chloroform ⁇ Mix well by vortexing until it becomes white ⁇ Centrifugation 12000g, 15 minutes, 4°C ⁇ Take only the aqueous layer (300 ⁇ l) ⁇ 100% ethanol 1.5 times the volume of the water layer taken (450 ⁇ l) ⁇ Add to column (add in 2 batches) ⁇ Centrifugation 8000g, 1 minute, 25°C ⁇ Buffer RWT 700
- a method for producing cells for dendritic cell therapy using a neoantigen possessed by a subject wherein monocytes are produced from peripheral blood mononuclear cells collected from the subject using apheresis. isolating, wherein the apheresis conditions for performing the apheresis include circulating tumor cell enrichment conditions; and circulating tumor cells from the isolated peripheral blood mononuclear cells.
- a step of isolating a step of extracting DNA and/or RNA from the blood-circulating tumor cells to create a DNA/RNA sequence library, a step of identifying a neoantigen based on the mutation information of the sequence library; introducing a neoantigen into a dendritic cell.
- dendritic cells may be obtained from a subject by apheresis or derived from monocytes isolated from peripheral blood mononuclear cells taken from the subject. You can also use things.
- Dendritic cells when producing dendritic cells from frozen raw materials, cryopreservation and transportation of apheresis raw materials, thawing, washing, and centrifugation of raw materials, supernatant after adhesion treatment, removal of floating cells, Dendritic cells can be used for dendritic cell therapy by performing dendritic cell induction, dendritic cell maturation/activation, and the like.
- monocytes are isolated from peripheral blood mononuclear cells collected from a subject using apheresis, CTCs are collected from PBMCs after monocyte removal, and the CTCs are used to Expressed neoantigen proteins can also be identified by proteomic analysis of CTCs.
- DNA can be extracted from CTCs and normal cells, exome analysis can be performed, and then neoantigens can be identified using software.
- Neoantigen proteins expressed in CTCs can be identified by proteome analysis of CTCs, and repertoire analysis and Elispot analysis can be used for analysis of peripheral blood T cells after administration of DCs.
- DCs By pulsing DCs with neoantigen peptides identified using CTCs, it is confirmed whether neoantigen-reactive T cells can be detected in the peripheral blood of cancer patients (Fig. 3).
- the proteome analysis can be performed by any method, and is not particularly limited as long as it can be analyzed using CTC. For example, the following method can be used. (1) About 1,000 to tens of thousands of CTCs in suspension are centrifuged at 1,000 to 2,000 cpm for 10 to 15 minutes to prepare a cell pellet. Cell pellets are stored in a -80°C cryogenic bath or liquid nitrogen container.
- protein is extracted from the CTC pellet by any one of detergent, freeze-thaw, and osmotic shock; (3) The extracted protein is subjected to a combination of a high performance liquid chromatograph (HPLC) and a triple quadrupole mass spectrometer (MS/MS) (liquid chromatograph mass spectrometer "Liquid Chromatograph-Mass Spectrometry: LC- MS/MS”). This identifies proteins expressed in CTCs.
- HPLC high performance liquid chromatograph
- MS/MS triple quadrupole mass spectrometer
- exome analysis can be performed using CTCs isolated as described above.
- Exome analysis can be analyzed by any method, and is not particularly limited as long as it can be analyzed using CTC, for example, it can be performed by the following methods.
- a library for whole exome sequence analysis is prepared from total DNA of CTCs and control cells using Agilent Technologies' SureSelect All Exon kit.
- a quality check of the generated sequence library is performed using an Agilent TapeStation.
- Sequencing is then performed on an Illumina NovaSeq 6000; (3) The reads obtained by sequencing are mapped using GEM (Marco-Sola, et al, 2012).
- the CTCs obtained as described above can be used for various analyses, other than neoantigen identification.
- a method of preparing a sample comprising circulating tumor cells from a subject for analysis without nucleic acid amplification and/or expansion by culture comprising: A step of isolating monocytes from peripheral blood mononuclear cells collected from a subject, wherein apheresis conditions for performing said apheresis include blood circulating tumor cell enrichment conditions; a step of isolating blood circulating tumor cells from the peripheral blood mononuclear cells; optionally, confirming the purity of the blood circulating tumor cells necessary for the analysis; and pre-treating circulating tumor cells for said analysis.
- a method for analyzing circulating tumor cells derived from a subject without nucleic acid amplification and/or proliferation by culture comprising: a step of isolating monocytes from nucleocytic cells, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; and monocytes from said isolated peripheral blood mononuclear cells.
- a method is provided comprising: isolating circulating tumor cells; and performing an analysis using the circulating tumor cells.
- such analysis is not particularly limited as long as it can be performed using CTC.
- whole exome analysis whole genome analysis, RNA-Seq, single-cell RNA-Seq, proteome Analysis can include transcriptome analysis.
- a sample comprising circulating tumor cells prepared by the methods of the present disclosure can be subjected to analysis as described above to identify neoantigens possessed by the subject.
- other analyses as appropriate, may employ the features described elsewhere herein.
- the circulating tumor cells can be appropriately pretreated depending on the intended analysis, and DNA, RNA, or protein can be extracted from the circulating tumor cells.
- DNA, RNA, or protein can be extracted from the circulating tumor cells.
- extraction means is not particularly limited, and known extraction techniques can be used as long as they can be extracted with an amount and accuracy that can be used for the intended analysis.
- the amount of DNA or RNA to be extracted should be an amount that can be used for subsequent analysis, for example, at least about 100 pg of DNA and/or RNA can be extracted.
- the extracted amount of DNA and/or RNA may be at least about 200 pg, about 500 pg, about 800 pg, about 1 ng, about 10 ng, about 30 ng, about 50 ng, about 80 ng, about 100 ng, about 200 ng. can.
- such DNA or RNA can be used to generate a DNA/RNA sequencing library.
- the neoantigen can be identified based on the mutation information of this sequence library.
- a method of treating or preventing a subject by analyzing circulating tumor cells without nucleic acid amplification and/or proliferation by culture wherein isolating monocytes from the isolated peripheral blood mononuclear cells, wherein apheresis conditions for performing said apheresis comprise circulating tumor cell enrichment conditions; and said peripheral blood from which monocytes have been isolated a step of isolating blood-circulating tumor cells from mononuclear cells, a step of analyzing using the blood-circulating tumor cells, and a step of treating or preventing the subject based on the analysis results.
- a method is provided, comprising:
- such methods of treatment or prevention may employ features described elsewhere herein as appropriate.
- neoantigen dendritic cells The procedure for producing neoantigen dendritic cells is described below (Fig. 1).
- PBMC peripheral blood mononuclear cells
- Spectra Optia Spectra Optia
- PBMCs contain monocytes, lymphocytes, circulating cancer cells (CTCs), etc., and hardly contain erythrocytes, granulocytes, and platelets.
- PBMC are stored in a storage bag attached to a centrifugal blood component separator together with a portion of plasma.
- apheresis conditions are shown to favor a spillover volume of about 9 to about 12 ml, and/or a buffy coat volume of about 7 to about 10 ml.
- the spillover volume was adjusted to 10 to 11 ml
- the buffy coat volume was set to 8 ml
- the other conditions were the default settings for collecting mononuclear cells in the apparatus.
- Example 2 Isolation of monocytes, the source of dendritic cells, from PBMC fractions
- (1) Separation and Seeding of PBMCs 20 ml of Ficoll is placed in multiple 50 ml centrifuge tubes. The solution (20-25 mL) containing PBMCs from the storage bag is layered over the Ficoll in the centrifuge tube. After centrifugation at 20° C., 500 G for 30 minutes, the buffy coat containing PBMC is collected with a pipette and placed in a 50 ml centrifuge tube. Add PBS, centrifuge, and remove the supernatant 3 to 5 times. Finally, AIM medium is added to make 40 ml. A small amount of AIM medium containing suspended PBMCs is taken, the cell count is calculated, and blood imaging is performed to measure the percentage of monocytes. Determine the total number of monocytes obtained from these numbers.
- a necessary amount of AIM medium is added to the AIM medium containing PBMCs suspended so that the final number of monocytes is 1 to 2 ⁇ 10 7 cells/6 ml per petri dish.
- 6 ml of the AIM medium containing the adjusted PBMC is seeded in each petri dish and placed in an incubator for 30 minutes. After confirming the attachment of cells to the bottom surface with a microscope, the supernatant is removed. Collect the supernatant containing floating cells in a centrifuge tube. After adding 6 ml of fresh AIM medium, the cells are placed in an incubator and cultured for 16 to 24 hours.
- the supernatant containing floating cells obtained in (1) contains CTCs and lymphocytes. Check the cell count and viability of this suspension. After the suspension is dispensed into centrifuge tubes, it is centrifuged to remove the supernatant. Add 1 ml of Cellbanker® to 1-3 ⁇ 10 7 cells and pipette. The cell suspension is aliquoted into cryotubes and stored frozen in a ⁇ 80° C. deep freezer until transported to the facility for CTC isolation.
- the immature dendritic cell suspension was centrifuged, the supernatant was discarded, and maturation medium (final concentration of 10 ⁇ g/ml picibanil, 50 ng/ml PGE2, AIM medium containing 20 ⁇ g/ml neoantigen peptide) is added. 6 to 12 ml of the cell suspension is placed in a petri dish and cultured in an incubator for 16 to 48 hours to induce mature dendritic cells.
- maturation medium final concentration of 10 ⁇ g/ml picibanil, 50 ng/ml PGE2, AIM medium containing 20 ⁇ g/ml neoantigen peptide
- the culture supernatant is used to pipette the culture surface of the petri dish of mature dendritic cells. After that, the culture medium containing the cells is collected in a centrifugation tube. 10 ml of AIM medium is added to the petri dish, and the culture surface is pipetted to collect the AIM medium in a centrifuge tube. After repeating this twice, the suspension of mature dendritic cells is centrifuged at 4° C., 500 G for 5 minutes and the supernatant is removed. Add 20 ml of AIM medium and collect in a centrifugal tube fitted with a cell strainer.
- this centrifugation tube After counting the number of cells, this centrifugation tube is centrifuged at 4° C., 500 G, 5 minutes, and the supernatant is removed. Suspend in a cell banker solution to a final cell concentration of 1 ⁇ 10 7 cells/ml and dispensed into cryotubes. The cryotubes are stored in a -80°C deep freezer or liquid nitrogen tank until the mature dendritic cells are inoculated into the patient.
- PBMC Mono- monocyte-free PBMC
- PBMC Mono- PBMC Mono- fraction
- PBMC Mono-fractions delivered in a frozen state are stored in deep freezers or liquid nitrogen tanks until analysis.
- Monoclonal antibodies used for CTC collection include anti-EpCAM antibody (epithelial cell marker), anti-Vimentin antibody, anti-N-Cadherin antibody (epithelial-mesenchymal transition, Mesenchymal Epithelial Transition: EMT marker), anti-CD20 antibody (malignant lymphoma marker) Select one or more appropriate for carcinoma or sarcoma, including CD45 antibody is used for control lymphocytes. Each antibody is labeled with a fluorescent substance including FITC and PE. Antibodies are applied to CTCs at a concentration of 5-10 ⁇ g/ml (0.1-100 ⁇ g). CTCs and control cells (CD45-positive cells) are then sorted using a cell sorter (SONY SH800).
- Example of sorting Number of cells to be sorted 1 ⁇ 10 7 cells PBS buffer (2 mM EDTA, 2% FBS)...485 ⁇ l CD45 PE (200 ⁇ g/ml) ..................10 ⁇ l EpCAM FITC (1 mg/ml) briefly........5 ⁇ l 500 ⁇ l
- the harvested cells are shown in Table 1 below and Figure 2.
- DNA extraction was performed as follows. DNA extraction with QIamp® DNA Mini Place 20 ⁇ l Proteinase K in a 1.5 ml microfuge tube. A sample suspended in 200 ⁇ l of PBS is added to this tube. Furthermore, 200 ⁇ l of Buffer AL is added and thoroughly mixed by vortexing. Incubate at 56° C. for 10 minutes, then spin down to collect solution on the inside of the lid. Add 200 ⁇ l of 100% ethanol and mix thoroughly by vortexing. Add the sample to the QIamp Mini Spin Column, close the lid and centrifuge at 6,000 g for 1 minute. Discard the filtrate and collection tube and attach a new collection tube. Add 500 ⁇ l Buffer AW1. Close the lid and centrifuge at 6,000 g for 1 minute.
- CTC DNA extracted from 4 cases DNA can also be extracted using NucleoSpin (registered trademark) Blood L (TAKARA).
- NucleoSpin registered trademark
- Blood L TAKARA
- Each collected cell group is centrifuged to remove the supernatant, and then suspended in 350 ⁇ l of RLT buffer solution (1 ml of Buffer RLT Plus (QIAGEN), 10 ⁇ l of ⁇ -ME (company name catalog number)).
- Example 4 Extraction of RNA from CTCs (RNeasy Mini Kit: QIAGEN)
- the RLT buffer solution containing CTCs and CD45+ cells was subjected to a gDNA Eliminator spin.
- 350 ⁇ l of 70% ethanol is added to the solution collected by applying to the column and centrifuging.
- 700 ⁇ l of buffer RW1 is added to the solution collected by centrifugation and applied to a Rneasy Min Elute spin column and centrifuged.
- 500 ⁇ l of buffer RPE, 500 ⁇ l of 80% ethanol, and 14 ⁇ l of Rnase-free water are used respectively in the same manner as RW1 to finally extract RNA.
- the amount of RNA is measured using NANO DROP ONE (Thermo SCIENTIFIC).
- RNA extraction is also possible using Kiazol and Trizol.
- DNA can also be extracted from CTCs by the following method.
- a library for whole exome sequence analysis is created from the total DNA of CTC and control cells using Agilent Technologies' SureSelect All Exon kit.
- a quality check of the generated sequence library is performed using an Agilent TapeStation. As a result of the quality inspection, confirm that the sequence library was produced without any problems.
- sequencing is performed by Illumina's NovaSeq 6000.
- Sequencing reads are mapped using GEM (Marco-Sola, et al, 2012). Mapping is also possible with Subread (Liao, et al., 2013), HISAT/HISAT2 (Kim, et al., 2015), or KART (Lin and Hsu, 2017). The number of reads mapped by HTSeq is then counted and gene expression analysis is performed. From the map results, the Genome Analysis Toolkit (GATK) "Haplottype Caller" (Version: 4.0.10.1) is used to make a variant call to create a vcf file.
- GEM Genome Analysis Toolkit
- Example 5 Sequencing RNA extracted from CTCs
- RNA sequence flow sample total RNA
- NEB rRNA depletion and TruSeq Stranded mRNA library Sequencing library construction
- HiSeq2500, 50-base paired-end Filtering of reads, trimming (moirai)
- Mapping STAR
- HTSeq,egdeR Variant call
- GATK Haplotype Caller Variant call
- Result create vcf file
- RNA-Seq library is prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit.
- the generated sequence library is quality-checked using an Agilent TapeStation and quantified by qPCR. As a result of the quality inspection, it is confirmed that the sequence library has been produced without problems, and a 5-plex RNA-Seq library is produced.
- Example 6 Identification of neoantigen in silico
- the vcf files of RNA sequences of CTC and control cells are used to identify neoantigens in silico and select those predicted to have T cell responses. Analysis uses software including NetMHC, MHCflurry, OpenVax, Neoantimon.
- the HLA class I and class II binding peptide structures of the selected neoantigen are determined, and the affinity for each HLA is predicted.
- Peptides with high affinity to HLA molecules are prepared (assigned to peptide synthesis companies such as Cosmo Bio Co., Ltd. and Takara Bio Co., Ltd.) and administered to dendritic cells in II (3).
- the peptides to be administered may be HLA class I peptides, HLA class I peptides plus class II peptides, or long peptides containing HLA class I and class II peptides.
- Neoantimon is an open program developed at the Institute of Medical Science, the University of Tokyo, and is software for identifying neoantigens specific to cancer cells in the environment (Mac/Linux).
- Mac/Linux As an input file, it is possible to use a vcf file created by a mutation analysis program. It is possible to comprehensively calculate HLA affinity and extract peptides (neoantigens) with high antigen display ability (https://github.com/hase62/Neoantimon).
- Tables 3 and 4 below show the results of identifying neoantigens in colon cancer cases.
- Somatic mutation (2) Identified neoantigens (SEQ ID NOs: 1-22 from top to bottom for sequences listed in column Neoantigen)
- Example 7 Flow until production of pulsed cells
- the neoantigen-DC manufacturing process flow is shown below.
- Freezing medium preparation For short peptides Five. Freezing medium preparation and dispensing of reagents 5.1. Freezing medium preparation 5.1.1. Based on the total number of cells in DCM, calculate the required amount of freezing medium and the amounts of medium, DMSO, and albumin to be used. 5.1.2. Weigh the calculated amount of medium into a centrifuge tube. 5.1.3. Add the calculated amount of DMSO to the measured medium and mix by inverting at least 5 times. 5.1.4. Add the calculated amount of albumin to 5.1.3. and mix by inverting at least 5 times. 5.1.5. Weigh an equal volume of the prepared freezing medium into a centrifuge tube. 5.1.6. Store the freezing medium and medium in a refrigerator set at 4°C until just before use.
- Physiological saline and distilled water injection Dispense only when using antigens that dissolve in physiological saline and distilled water. However, this operation is not necessary when using a dispensed item. 5.3.1. Clean the caps of the saline and distilled water bottles with an alcohol swab and open them. 5.3.2. Weigh any amount into a sample tube. 5.3.2. Store in a refrigerator set at 4°C until just before use.
- Cell suspension 1 Measure the liquid volume to calculate the number of cells. 6.2.5. After mixing by inverting at least 5 times, collect 50 ⁇ L in a microtube using a micropipette. 6.2.6. Dilute with trypan blue staining solution 2 to 20 times, and count the number of viable and dead cells using a hemocytometer. 6.2.7. Calculate the total number of viable cells from the counts. 6.2.8. Calculate the amounts of medium and antigen to be used based on the calculated total number of cells. 6.2.9. When pulsing the antigen, dissolve it in the specified solvent to a concentration of 2 mg/mL. 6.2.10. Weigh the calculated amount of medium into a centrifuge tube.
- 6.2.11 Withdraw approximately 5 mL from each aliquot of medium and dissolve the calculated amount of each antigen.
- 6.2.12. Pass 6.2.11 through a syringe filter and add to the medium in 6.2.10 (hereinafter referred to as "peptide-supplemented medium").
- 6.2.13. Dispense cell suspension 1 into centrifugation tubes according to the number of types of antigens to be used, and centrifuge. Setting conditions: 500G 5min 4°C 6.2.14. After centrifugation, collect the supernatant in a 50 mL test tube. 6.2.15.
- tube 1 Using a pipette, combine the cells from each centrifuge tube with 5-20 mL of medium into one 50 mL centrifuge tube (hereinafter "tube 1"). 6.1.12. Wash each centrifugal tube after collecting the cells with the culture medium and combine them in centrifugal tube 1, and dilute up to about 40 mL (hereinafter referred to as "cell suspension 1"). * Measure the liquid volume to calculate the number of cells.
- Neoantigen peptide-pulsed dendritic cells are obtained as described above.
- ⁇ Final formulation process ⁇ Preparation of cell density (2 ⁇ 10 7 cells/mL) Cells are suspended in medium. ⁇ Mixing with cell cryopreservation solution (1 ⁇ 10 7 cells/mL) Mix the cell suspension (medium) and the cell cryopreservation solution at a ratio of 1:1. The prepared concentration is concentrated (2-fold) so that it is diluted 2-fold at the time of use. Cells of 0.1 to 1 ⁇ 10 7 cells/mL can be cryopreserved at 0.5 to 1 mL/tube. ⁇ Filling into a cryotube etc. (1 ⁇ 10 7 cells/1 mL/tube) Dispense the cell suspension into cryotubes.
- cryopreserved cells ⁇ -80°C
- Normal saline room temperature
- Example 8 Dendritic cell therapy
- Dendritic cell therapy is performed using the pulsed dendritic cells produced in Example 7.
- a solution thawing solution
- a cryovial containing DCs (1 ⁇ 10 7 /1 mL) frozen under liquid nitrogen is thawed to about 70% using a heat block heated to 37°C.
- 3) Pour a portion of the thawing solution into a centrifuge tube, and while co-washing the cryovial containing the DC with the rest of the thawing solution, transfer it to the centrifuge tube (total volume: 10 ml).
- proteome analysis In this example, after collecting CTCs, neoantigen proteins expressed in CTCs are identified by proteome analysis of CTCs.
- the method of proteome analysis is as follows. (1) About 1,000 to tens of thousands of CTCs in suspension are centrifuged at 1,000 to 2,000 cpm for 10 to 15 minutes to prepare a cell pellet. Cell pellets are stored in a -80°C cryogenic bath or liquid nitrogen container.
- protein is extracted from the CTC pellet by any one of detergent, freeze-thaw, and osmotic shock; (3) The extracted protein is subjected to a combination of a high performance liquid chromatograph (HPLC) and a triple quadrupole mass spectrometer (MS/MS) (liquid chromatograph mass spectrometer "Liquid Chromatograph-Mass Spectrometry: LC- MS/MS”). This identifies proteins expressed in CTCs.
- HPLC high performance liquid chromatograph
- MS/MS triple quadrupole mass spectrometer
- Example 10 Isolation of CTCs from various cancer patients
- CTCs were isolated using peripheral blood mononuclear cells from various cancer patients.
- lung cancer stage 4
- colon cancer stage 4
- ovarian cancer stage 4
- duodenal papilla cancer stage 4
- colon cancer stage 4
- liver PBMCs derived from cancer, multiple liver cancer, prostate cancer (stage 4), and esophageal cancer patients were used.
- the methods of collection of PBMCs, isolation of monocytes from PBMCs, and isolation of CTCs were performed in the same manner as in Examples 1-3.
- the results are shown in Figures 4-9.
- the flow cytometry analysis results shown in FIGS. 4 to 6 are for cases with a large number of CTCs, and the flow cytometry analysis results shown in FIGS. 7 to 9 are for cases with a small number of CTCs. Cases with a small number of CTCs are cases in which the treatment has been successful or in which the cancer has been completely resected by surgery.
- RNA sequencing or identification of neoantigens by exome analysis and proteome analysis
- Neoantigens were identified by RNA sequencing or exome analysis using CTCs isolated from various cancer patients. The results are shown in Tables 5-13 below.
- RNA sequencing was performed in the same manner as in Example 5.
- Tables 5-8 are the results of neoantigen identification by exome analysis. By adding proteome analysis, it becomes possible to narrow down and identify only neoantigens expressed as proteins. Narrowing down identification of neoantigens is also possible by combining RNA sequencing with exome analysis instead of proteome analysis.
- Tables 9-12 are the results of neoantigen identification by RNA sequencing.
- Neoantigens SEQ ID NOS: 23-38, top to bottom, for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOs: 39-48 from top to bottom for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOS: 49-64, top to bottom, for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOs: 65-71 from top to bottom for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOS: 72-78 from top to bottom for the sequences listed in column Neoantigen
- Neoantigens SEQ ID NOs: 79-85 from top to bottom for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOs: 86-96 from top to bottom for sequences listed in column Neoantigen
- Neoantigens SEQ ID NOs: 97-98 from top to bottom for sequences listed in column Neoantigen
- Example 12 Other analysis examples using CTC-disease other than cancer
- CTC gene analysis reveals various gene mutations including driver genes, enabling the selection of molecular-targeted drugs.
- MSI-High high-frequency microsatellite instability
- (3) By combining transcriptome analysis, single-cell RNA-seq, and clinical data, the differences in CTC between metastatic (or susceptible) and non-metastatic (or unlikely) cases are clarified. can do. This will lead to prognosis prediction of cases and elucidation of the mechanism of metastasis.
- metabolome analysis refers to comprehensive detection of all types and concentrations of metabolites present in a target sample and analysis of the results; also known as metabolomics); , clarifies the metabolic properties of CTCs, and makes it possible to examine (3) more comprehensively.
- Example 13 Other analysis examples using CTC-Confirmation of health condition
- CTC exome analysis can detect whether a malignant tumor is caused by a rare variant mutation. Furthermore, the presence of the same disease may be revealed by testing relatives, and early diagnosis and early treatment of relatives becomes possible.
- DNA of normal cells can be extracted in addition to CTCs when identifying neoantigens. By whole genome analysis or DNA chip analysis of this normal DNA, the genome sequence of the normal germline can be determined. Based on this sequence, it is possible to calculate the risk of not only rare variant mutations related to diseases but also common variants. It is also useful for preventive medicine against diseases other than malignant tumors.
- the method of the present disclosure can noninvasively identify a patient's neoantigen and is useful for clinical trials of cancer immunotherapy using neoantigen. It is also expected to expand into regenerative medicine and develop new medical treatments centered on cell therapy.
- SEQ ID Nos: 1-22 Neoantigens identified in Example 6
- SEQ ID Nos: 23-98 Neoantigens identified in Example 11
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Oncology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
(項目X1)
核酸増幅及び/または培養による増殖をせずに解析するための被験者由来の血中循環腫瘍細胞を含む試料を調製する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
必要に応じて、該解析に必要な血中循環腫瘍細胞の純度を確認する工程と、
必要に応じて、該血中循環腫瘍細胞に対して、該解析のための前処理を行う工程と
を含む、方法。
(項目X2)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目に記載の方法。
(項目X3)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目X4)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目X5)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目X6)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目X7)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目X8)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目X9)
前記解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、上記項目のいずれか一項に記載の方法。
(項目X10)
前記試料が、前記解析を行うことによって前記被験者が有するネオアンチゲンを同定するためのものである、上記項目のいずれか一項に記載の方法。
(項目X11)
さらに、前記血中循環腫瘍細胞からDNA、RNA、またはタンパク質を抽出する工程を含む、上記項目のいずれか一項に記載の方法。
(項目X12)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目X13)
核酸増幅及び/または培養による増殖をせずに被験者由来の血中循環腫瘍細胞を解析する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞を用いて解析を行う工程と
を含む、方法。
(項目X14)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目のいずれか一項に記載の方法。
(項目X15)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目X16)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目X17)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目X18)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目X19)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目X20)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目X21)
該解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、上記項目のいずれか一項に記載の方法。
(項目X22)
上記項目のいずれか一項に記載の方法によって前記被験者由来の前記血中循環腫瘍細胞を解析することにより、前記被験者が有するネオアンチゲンを同定する方法。
(項目X23)
さらに、前記血中循環腫瘍細胞からDNA、RNA、またはタンパク質を抽出する工程を含む、上記項目のいずれか一項に記載の方法。
(項目X24)
さらに、前記DNAまたはRNAからDNA/RNAシーケンスライブラリを作成する工程と、
前記シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と
を含み、前記血中循環腫瘍細胞の純度が少なくとも約20%である、上記項目のいずれか一項に記載の方法。
(項目X25)
前記解析が、前記DNAの全エクソーム解析を含む、上記項目のいずれか一項に記載の方法。
(項目X26)
前記解析が、さらに、前記血中循環腫瘍細胞のプロテオーム解析を含む、上記項目のいずれか一項に記載の方法。
(項目X27)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目X28)
被験者が有するネオアンチゲンを用いた樹状細胞療法のための細胞を製造する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞からDNAまたはRNAを抽出し、DNA/RNAシーケンスライブラリを作成する工程と、
該シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と、
該ネオアンチゲンを樹状細胞に導入する工程と
を含む、方法。
(項目X29)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目のいずれか一項に記載の方法。
(項目X30)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目X31)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目X32)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目X33)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目X34)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目X35)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目X36)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目X37)
前記樹状細胞は、前記アフェレーシスによって前記被験者から得られたものであるか、または前記単球から誘導されるものである、上記項目のいずれか一項に記載の方法。
(項目X38)
核酸増幅及び/または培養による増殖をせずに血中循環腫瘍細胞を解析することにより被験者を治療または予防する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞を用いて解析を行う工程と、
該解析結果に基づいて、該被験者を治療または予防する工程と
を含む、方法。
(項目X39)
該解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、上記項目のいずれか一項に記載の方法。
(項目1)
核酸増幅及び/または培養による増殖をせずに配列決定することにより、被験者が有するネオアンチゲンを同定するための試料を調製する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
必要に応じて、ネオアンチゲンの同定に必要な血中循環腫瘍細胞の純度を確認する工程と、
該血中循環腫瘍細胞を、ネオアンチゲンを同定するための試料として提供し、及び/または該血中循環腫瘍細胞からDNA及び/またはRNAを抽出する工程と
を含む、方法。
(項目2)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目に記載の方法。
(項目3)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目4)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目5)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目6)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目7)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目8)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目9)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目A1)
核酸増幅及び/または培養による増殖をせずに配列決定することにより、被験者が有するネオアンチゲンを同定するための試料を調製する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
必要に応じて、ネオアンチゲンの同定に必要な血中循環腫瘍細胞の純度を確認する工程と、
該血中循環腫瘍細胞を、ネオアンチゲンを同定するための試料として提供し、及び/または該血中循環腫瘍細胞からDNA及び/またはRNAを抽出する工程と
を含む、方法。
(項目B1)
核酸増幅及び/または培養による増殖をせずに配列決定することにより、被験者が有するネオアンチゲンを同定する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞からDNA及び/またはRNAを抽出し、DNA/RNAシーケンスライブラリを作成する工程と、
該シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と
を含み、該血中循環腫瘍細胞の純度が少なくとも約20%である、方法。
(項目B2)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目のいずれか一項に記載の方法。
(項目B3)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目B4)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目B5)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目B6)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目B7)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目B8)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目B9)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目C1)
被験者が有するネオアンチゲンを用いた樹状細胞療法のための細胞を製造する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞からDNA及び/またはRNAを抽出し、DNA/RNAシーケンスライブラリを作成する工程と、
該シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と、
該ネオアンチゲンを樹状細胞に導入する工程と
を含む、方法。
(項目C2)
前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、上記項目のいずれか一項に記載の方法。
(項目C3)
前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、上記項目のいずれか一項に記載の方法。
(項目C4)
前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、上記項目のいずれか一項に記載の方法。
(項目C5)
前記血中循環腫瘍細胞が約1×103個以上である、上記項目のいずれか一項に記載の方法。
(項目C6)
前記末梢血単核球細胞が約1×104個以上である、上記項目のいずれか一項に記載の方法。
(項目C7)
前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、上記項目のいずれか一項に記載の方法。
(項目C8)
さらに、前記血中循環腫瘍細胞を凍結する工程を含む、上記項目のいずれか一項に記載の方法。
(項目C9)
前記DNAまたはRNAが、少なくとも約100pg抽出される、上記項目のいずれか一項に記載の方法。
(項目C10)
前記樹状細胞は、前記アフェレーシスによって前記被験者から得られたものであるか、または前記単球から誘導されるものである、上記項目のいずれか一項に記載の方法。
以下に本開示の好ましい実施形態を説明する。以下に提供される実施形態は、本開示のよりよい理解のために提供されるものであり、本開示の範囲は以下の記載に限定されるべきでない。したがって、当業者は、本明細書中の記載を参酌して、本開示の範囲内で適宜改変を行うことができることは明らかである。また、本開示の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができる。
内側の血漿分画との境界)が光学的センサーにより自動的に検出される。
1.遠心器
インナーローター回転速度:300~2200rpm
アウターローター回転速度:150~1100rpm
最高回転速度:2200rpm
2.ポンプ
形式:ラインローラーポンプ
速度範囲:0.3~21rpm(採取ポンプ、血漿ポンプ、全血ポンプ、
ACDポンプ/循環ポンプ)
1~28rpm(ACDポンプ)
流速範囲 採血:12.4~120mL/分
血漿:5.12~80mL/分
採取:1.78~80mL/分
抗凝固剤:1.78~17.5mL/分
3.プログラムの種類
Plt-5d:両針法による5日間までの保存のための血小板の採取
Plt-5d-SN:単針法による5日間までの保存のための血小板の採取
PBSC Lymphocyte:末梢血幹細胞やリンパ球の分離
RV-PBSC:少量に濃縮された末梢血幹細胞の採取
BMSC:骨髄からの幹細胞の分離
Granulocyte:顆粒球の採取
MNC:単核球の採取
除去:血漿又は血漿とリンパ球の治療的除去
TPE:治療的血漿交換
吸着:治療的血漿分離
RBC:治療的赤血球交換及び除去
まず装置の電源を入れ、専用ディスポーザブル血液回路(アフェレーシスセット)を装着する。分離チャンバーをチャンバーホルダーに装着した後、プライミングを実施する。一実施形態において、血液成分の分離は以下のようにして行うことができる。
(1)生食ラインを完全に脱気し、ディスプレイに従って分離前の準備を行う。
(2)(Continue)キーを押して、採血針を供血者に穿刺し、供血前サンプルを採取する。返血針を穿刺する。
(3)患者情報を入力する
(4)(Start)キーを押して分離を開始する。分離及び採血は自動的に行われ、目標設定値に達すると自動的に終了する。
(1)(Continue)キーを押して、生食ラインのクランプを開放し、供血者から採血ラインを外して返血前の準備を行う。(単針法の場合は採血ラインを外さない。)
(2)(Start)キーを押し、返血を開始する。返血は自動的に行われ、終了する。(3)返血終了後、(Stop)キーを押す。
まず装置の電源を入れ、装置に血液回路を装着させ、回路のテストおよびプライミングを行う。その後、画面の指示に従って患者/ドナーを接続し、採血ラインと返血ラインのクランプを開き、血液成分の分離を行う。
(1)回路に付属の採血ラインに導入された患者/ドナーの全血は採血ポンプの働きにより、遠心分離器に装着された回路内のチャネル部に導入される。
(2)血液が凝固しないように一定比率で抗凝固剤が使用される。抗凝固された血液が、患者/ドナーへ返血される。
(3)遠心分離器の回転とともに回転してチャネル内の血液が成分毎に分離される。
(4)遠心分離を経た各成分は、血漿、血小板、赤血球の3ラインのチューブに分かれて遠心分離器より流出し、ポンプ動作により回路内のカセット部に入る。その後、バルブ動作により分岐部を経て、採取バッグ、又はカセット部に構成される返血リザーバーのいずれかに導入される。
(5)返血リザーバーに導かれた血液は返血ポンプの働きにより、返血ラインを経て患者/ドナーに返血される。尚、Exchangeセットにおいては、置換液が置換液/採取ポンプで、置換液ラインを通じてカセット内のリザーバーに導入され、患者に供給される。
・PCモニター、キーボード、マウス
・コンプレッサー
・本体
・PC
*本体のタンクに水が入っているか確認する。
<PC画面>
PW : fcm
デスクトップのソフト起動
↓
チップを登録(100μm)
↓
NEXT→PUSH OPEN
↓
チップのドアオープン、古いチップがあれば捨てる
↓
CHIP INSERTにチップ挿入 (文字が正面になる向き)
↓
NEXT
↓
レーザー設定、フィルター設定
↓
NEXT
↓
このままで少し待つ。セッティングが完了したら、succesfullyになる。→キャリブレーション(5mlチューブにビーズを入れる)
↓
本体にビーズをセットして、Start (standard、□にはチェック入れない)
↓
このままで少し待つ。キャリブレーションが完了したら、succesfullyになる。
↓
OK
↓
テンプレートを開く(CD45, EpCAMのテンプレート)
↓
Create new experiment
↓
少し流す。Sample puressure 5くらいで少し流して止める。
↓
流した画面を確認して、ゲートをかけてどこを採るか設定する。
↓
L,Rそれぞれに、採りたい区画と数を入力
Method:2Way、Mode:Normal
↓
採ってきた細胞を入れるチューブをセットしてドアを閉める(15mlチューブにCML6mlくらい)。
↓
Load Collection
↓
チューブが奥にセットされる。
↓
Experimentのファイル
↓
名前変更する
↓
流すサンプルをセット
↓
Start
↓
安定してきたら、Sort startする。その際に、recordもスタートする
↓
採りたい数まで到達した方はsortが止まる。サンプルが残り少なくなったら止める。
凍結細胞を37℃で溶かす
↓
フィルター ファルコン 凝集細胞を除く
↓
3ml PBS Buffer (2mM EDTA,2%FBS)
↓
遠心 1000rpm、2分、4℃
↓
細胞計数→遠心 1000rpm、2分、4℃
↓
抗体による染色
抗体濃度
CD45 PE 5μg/ml
EpCAM 10μg/ml
↓
氷上に30分
↓
2ml PBS Buffer(2mM EDTA,2%FBS)添加
↓
遠心 1000rpm、2分、4℃
↓
1ml PBS Buffer(2mM EDTA,2%FBS)に懸濁
↓
フィルター、Tube
↓
CML 6ml
CML
1% MEM Non-Essential Amino Acis Solution1% HEPES
1% Sodium Pyruvate
50μM 2-mercaptoethanol
1% Penicillin-Streptomycin
10% FCS(FBS)
500ml RPMI 1640 medium
↓
ソーティング(CD45 PE,EpCAM FITC)
↓
遠心 1800rpm、10分、4℃
↓
懸濁 RLT Buffer 350μl
RLT Buffer:
Buffer RLT Plus (QIAGEN) 1ml
β-ME 10μl
QIamp(登録商標)DNA MiniによるDNA抽出
20μl Proteinase K ←1.5ml マイクロチューブ
↓
PBS200μlで懸濁したサンプルをこのチューブに添加
↓
200μl Buffer ALを添加 ボルテックスで十分に混和
↓
56 ℃ 10分 インキュベート
↓
スピンダウンしてフタの内側についた溶液を回収
↓
100% エタノール200μl添加 ボルテックスで十分に混和
↓
QIamp Mini Spin Columnにサンプルを添加
↓
フタを閉めて遠心 6,000g, 1分
↓
ろ液とコレクションチューブは捨て、新しいコレクションチューブを付ける
↓
500μl Buffer AW1 添加
↓
フタを閉めて遠心 6,000g, 1分
↓
500μl Buffer AW2 添加
↓
フタを閉めて遠心 12,000g、3分
↓
ろ液を捨てて遠心 12,000g、1分
↓
QIamp Mini Spin Columnを新しい1.5ml マイクロチューブに移す
100μl Buffer AEを添加、室温で1分 インキュベート
↓
遠心 6,000g,1分
O.D測定し、抽出したDNAは-80℃ 保存
NucleoSpin(登録商標)Blood L(TAKARA)によるDNA抽出
Proteinase K: Proteinase Buffer PBを加えて溶かす(-20℃で保存)
Wash Buffer B5:100%エタノールを加える
↓
サンプル(最大2×107 cells)細胞(ペレット状)を用いる
↓
PBS 2mlで溶かす
↓
150μl Proteinase Kを入れる
↓
2ml BQ1 激しくボルテックス(10秒)
↓
56℃ でインキュベーション15分 ←ヒートブロックはあらかじめ温めておく
↓
2ml 100%エタノールを入れ、混ぜる
↓
DNA結合
NucleoSpin Blood L Column 3mlをサンプルにロード1回目 4,500g遠心,3分,25℃
↓
NucleoSpin Blood L Column 3mlをサンプルにロード2回目 4,500g遠心,5分,25℃
↓
洗浄1回目
2ml BQ2 4,500g遠心, 2分, 25℃
↓
洗浄2回目、Dry membrane
2ml BQ2 4,500g遠心, 10分, 25℃
↓
DNA抽出 新しい15mlチューブ ←Elution Buffer EBを70℃に温めておく
200μl Buffer BE (70℃に予熱したもの) 2分, インキュベーション,室温
4,500g遠心, 2分,25℃
↓
抽出したDNAは-80℃で保存
↓
遠心 10000g、1分、25℃
↓
遠心で落ちた液に70%エタノール350μl添加
↓
その全量をRneasy Min Elute spin columに添加
↓
遠心 10000g、1分、25℃
↓
Buffer RW1 700μl
↓
遠心 10000g、1分、25℃
↓
Buffer RPE 500μl
↓
遠心 10000g、1分、25℃
↓
80%エタノール 500μl
↓
遠心 10000g、2分、25℃
↓
乾燥 フタを開けて遠心 12000g、5分、25℃
↓
Rnase-free water 14μl
↓
遠心 12000g、1分、25℃
↓
定量(NANO DROP ONE, Thermo SCIENTIFIC)
凍結細胞を溶かす
↓
PBS(2% FBS,2mM EDTA)3mlに懸濁
↓
遠心 1000rpm、2分、4℃
↓
細胞数計測 1.91×106細胞
↓
500μlずつ2本に分ける(15mlチューブ)
↓
遠心 1000rpm、2分、4℃
↓
QIAzol Lysis reagent 700μl
TRIzol Reagent 700μl
それぞれに懸濁する
↓
クロロホルム 140μl
↓
ボルテックス 白くなるまでよく混ぜる
↓
遠心 12000g、15分、4℃
↓
水層だけ取る(300μl)
↓
100% エタノール 取った水層の1.5倍Volume(450μl)
↓
カラムに添加(2回に分けて添加)
↓
遠心 8000g、1分、25℃
↓
Buffer RWT 700μl
↓
遠心 8000g、1分、25℃
↓
Buffer RPE 500μl
↓
遠心 8000g、1分、25℃
↓
80% エタノール500μl
↓
遠心 8000g、2分、25℃
↓
乾燥 12000g、5分、25℃
↓
エリューション Rnase free Buffer 14μl(CTC20-001→20μlでエリューション)
↓
室温で2~3分おく
↓
遠心 12000g、1分、25℃
樹状細胞誘導、樹状細胞成熟化・活性化などを行うことにより、樹状細胞療法のための樹状細胞とすることができる。
(1)約1000個から数万個のCTC浮遊液を1000~2000cpmで10~15分遠心し細胞ペレットを作製する。細胞ペレットは-80℃の低温槽あるいは液体窒素容器で保存する。
(2)界面活性剤、凍結融解、浸透圧ショック、のいずれかの方法でCTCのペレットからタンパク質を抽出する。
(3)抽出したタンパク質を、高速液体クロマトグラフ(HPLC)と三連四重極型質量分析計(MS/MS)を組合せた装置(液体クロマトグラフ質量分析計「Liquid Chromatograph-Mass Spectrometry : LC-MS/MS」)で分析する。これによりCTCに発現しているタンパク質を同定する。
(1)CTC及びコントロール細胞のtotal DNAからAgilent technologies社のSureSelect All Exonキットを用いて、全エクソームシーケンス解析用ライブラリを作製する。作製されたシーケンスライブラリの品質検査を、Agilent社TapeStationを用いて実施する。
(2)次いでシーケンシングをイルミナ社NovaSeq 6000により行う。
(3)シーケンシングで得られたリードを、GEM(Marco-Sola、et al、2012)を用いてマッピングする。マッピングはSubread(Liao、et al、 2013)、HISAT/HISAT2(Kim、et al、2015)、あるいはKART(Lin and Hsu、2017)でも可能である。次にHTSeqによりマップされたリード数をカウントし、遺伝子発現解析を実施する。マップ結果からThe Genome Analysis Toolkit (GATK)“HaplotypeCaller”(Version:4.0.10.1)を用いてバリアントコールし、vcfファイルを作成する。
シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定することができる。
本明細書において引用された、科学文献、特許、特許出願などの参考文献は、その全体が、各々具体的に記載されたのと同じ程度に本明細書において参考として援用される。
アフェレーシスのための遠心型血液成分分離装置としては、コムテック(COM.TEC)(登録商標)(フレゼニウスカービ)やスペクトラ オプティア(テルモ)等などがある。
(1)PBMCの分離と播種
複数の50mlの遠心管にフィコールを20ml入れておく。保存用バッグのPBMCを含む溶液(20~25mL)を遠心管のフィコールに重層する。20℃,500G,30分で遠心し、PBMCが含まれるバフィーコートをピペットで採取して50ml遠心管に入れる。PBSを加えて遠心、上清除去を3から5回繰り返す。最後にAIM培地を入れ40mlにする。PBMCをサスペンドしたAIM培地を少量取り、細胞数を算出し、また血液像検査を行い単球の割合を計測する。これらの数値から得られた単球の総数を求める。
(1)で得られた浮遊細胞を含む上清にはCTCやリンパ球が存在する。この浮遊液の細胞数や生存率を確認する。浮遊液を遠心管に分注した後、遠心分離して上清を取り除く。1~3×107個の細胞に対し1mlのセルバンカー(登録商標)を加えピペッティングする。細胞懸濁液をクライオチューブに分注し、CTCを単離する施設に輸送するまで-80℃のディープフリーザーで凍結保存する。
インキュベーターからシャーレを取り出し、培養上清を用いて培養面を洗浄する。洗浄後、洗浄液を除去し、10mLのAIM培地またはPBSで洗浄し上清を除去する。最終濃度が50ng/mlのIL-4、50ng/mlのGM-CSFを含有するAIM培地をシャーレ1枚につき6~8mLずつ加える。その後インキュベーターに入れ、未熟樹状細胞誘導のため5日間培養する。
培養上清を用いて成熟樹状細胞のシャーレの培養面をピペッティングする。その後、細胞が含まれた培養液を遠心管に回収する。シャーレに10mlのAIM培地を加え培養面をピペッティングしてAIM培地を遠心管に回収する。これを2回繰り返した後、成熟樹状細胞の懸濁液を4℃,500G,5分遠心して上清を除去する。20mlのAIM培地を加え、セルストレーナーをセットした遠心管に回収する。細胞数を計測した後、この遠心管を4℃,500G,5分で遠心し、上清を除去する。最終細胞濃度が1×107個/mlになるようにセルバンカー溶液にサスペンドし、クライオチューブに分注する。成熟樹状細胞を患者に接種するまで、クライオチューブは-80℃のディープフリーザー、あるいは液体窒素タンクで保存する。
凍結保存した単球を含まないPBMC(PBMC Mono-)分画を、ドライアイスを入れたシッパーで解析施設に速やかに搬送する。解析施設では凍結状態で搬送されたPBMC Mono-分画を解析するまでディープフリーザーあるいは液体窒素タンクで保存する。
を含めた、癌腫あるいは肉腫に適切なものを1つ以上選択する。対照のリンパ球についてはCD45抗体を用いる。それぞれの抗体はFITCやPEを含む蛍光物質でラベルしておく。抗体は5~10μg/ml(0.1~100μg)の濃度でCTCに作用させる。その後セルソーター(SONY SH800)を用いてCTCとコントロール細胞(CD45陽性細胞)をソーティングする。
ソート元細胞数:1×107細胞
PBS Buffer(2mM EDTA,2% FBS)…485μl
CD45 PE(200μg/ml)……………………………10μl
EpCAM FITC (1mg/ml)………………………5μl
500μl
QIamp(登録商標) DNA MiniによるDNA抽出
20μl Proteinase Kを1.5mlマイクロチューブに入れる。PBS200μlで懸濁したサンプルをこのチューブに添加する。さらに200μl Buffer ALを添加しボルテックスで十分に混和する。56℃で10分間インキュベートし、その後スピンダウンしてフタの内側についた溶液を回収する。100% エタノールを200μl添加し、ボルテックスで十分に混和する。QIamp Mini Spin Columnにサンプルを添加し、フタを閉めて6,000gで1分間遠心する。ろ液とコレクションチューブは捨て、新しいコレクションチューブをつける。500μl Buffer AW1を添加する。フタを閉めて6,000gで1分間遠心する。500μl Buffer AW2を添加する。フタを閉めて12,000gで3分間遠心する。ろ液を捨てて12,000gで1分間遠心する。QIamp Mini Spin Columnを新しい1.5ml マイクロチューブに移す。100μl Buffer AEを添加して室温で1分間インキュベートする。6,000gで1分間遠心する。O.Dを測定し、抽出したDNAを-80℃で保存する。
CTCおよびCD45+細胞が入ったRLTバッファー溶液をgDNA Eliminator spin
columnに作用させ、遠心して採取した溶液に350μlの70%エタノールを添加する。その後Rneasy Min Elute spin columnに作用させ、遠心して採取した溶液にバッファーRW1 700μl添加遠心する。その後、バッファーRPE 500μl、80%エタノール 500μl、Rnase-free water 14μlでそれぞれRW1と同様の処理を行い、最終的にRNAを抽出する。RNA量はNANO DROP ONE(Thermo SCIENTIFIC)を用いて測定する。
サンプル(total DNA)
↓
エクソン領域の濃縮化、シーケンスライブラリ作製(Agilent technologies社SureSelect All Exon、IDT xGen社エクソームキット)
↓
シーケンシング(NovaSeq 6000システム、イルミナ社)
↓
マッピング(GEM、Subread、HISAT / HISAT2、KART)
↓
バリアントコール(GATAC)
↓
結果(vcfファイル作成)
・RNAのシーケンスの流れ
サンプル(total RNA)
↓
シーケンスライブラリ作製(NEB rRNA depletion and TruSeq Stranded mRNA library)
↓
シーケンシング(HiSeq2500, 50-base paired-end)
↓
リードのフィルタリング、トリミング(moirai)
↓
マッピング(STAR)
↓
マッピング(HTSeq,egdeR)
↓
バリアントコール(GATK HaplotypeCaller)
↓
結果(vcfファイル作成)
されたシーケンスライブラリの品質検査をAgilent社TapeStationを用いて実施し、qPCRにより定量する。品質検査の結果、シーケンスライブラリが問題なく作製されたことを確認し、5-plex RNA-Seqライブラリを作製する。
リアントコールし、vcfファイルを作成する。
CTCとコントロール細胞のRNAシーケンスのvcfファイルによりin silicoでネオアンチゲンを同定し、その中からT細胞の反応があると予測されるものを選択する。解析にはNetMHC、MHCflurry、OpenVax、Neoantimonを含むソフトウエアを使用する。選択されたネオアンチゲンのHLAクラスI、及びクラスIIへの結合ペプチド構造を決定し、それぞれのHLAに対する親和性を予測する。HLA分子への親和性が高いペプチドを作製し(コスモバイオ株式会社、タカラバイオ株式会社などのペプチド合成会社に依頼)、IIの(3)の樹状細胞に投与する。投与するペプチドはHLAクラスIペプチド、HLAクラスIペプチド+クラスIIペプチド、あるいはHLAクラスIとクラスIIを含むロングペプチドのいずれでも良い。
ネオアンチゲン-DC製造工程フローを以下に示す。
5.凍結培地調製及び試薬類分注
5.1.凍結培地調製
5.1.1. DCMの総細胞数を基に凍結培地の必要量及び培地、DMSO、アルブミンの各使用量を算定する。
5.1.2. 遠心管に算定量の培地を量り取る。
5.1.3. 量り取った培地に算定量のDMSOを加え、5回以上転倒混和する。
5.1.4. 5.1.3.に算定量のアルブミンを加え、5回以上転倒混和する。
5.1.5. 調製した凍結培地と等量の培地を遠心管に量りとる。
5.1.6. 凍結培地及び培地を使用直前まで4℃設定の保冷庫で保管する。
DMSOで溶解する抗原を使用する場合のみDMSOの分注を行う。ただし、既にDMSO溶解済みの抗原(分注したもの等)を同時に使用する場合は本操作は不要。
5.2.1. 注射針を装着したシリンジを用いて、DMSOを0.5mL程度サンプルチューブに量り取る。
5.2.2. 使用直前まで室温で保管する。
生理食塩水及び蒸留水で溶解する抗原を使用する場合のみ分注を行う。ただし分注品を使用する場合、本操作は不要。
5.3.1. 生理食塩水及び蒸留水溶のボトルの蓋をアルコール綿で清拭し開封する。
5.3.2. 任意の量をサンプルチューブに量り取る。
5.3.2. 使用直前まで4℃設定の保冷庫で保管する。
6.1. 成熟樹状細胞回収
6.1.1. DCM終了からの経過時間が12時間以上36時間以内である事を確認する。
6.1.2. インキュベータからシャーレを取り出し、作業エリアに搬入する。
※シャーレの内1枚、又は観察用シャーレを検鏡する。検鏡した観察用シャーレは破棄する。
※シャーレは原則5枚1組として扱い、1組毎に全てのシャーレに対して下記6.1.3.~6.1.8.の操作を行う。
6.1.3. ピペットを用いて、シャーレ内の細胞を培養上清ごと50mL遠心管に回収する。
DCM時に各抗原でパルスを実施した細胞と、実施していない細胞で遠心管を分ける。
(以下作業、各細胞毎に遠心管を分け、混合しないようにする。)
6.1.4. ペットを用いて、5~10mLの培地でシャーレ底面を満遍なく洗浄し、50mL遠心管に回収する。
6.1.5. ピペットを用いて、シャーレに培地を1~2mL分注する。
6.1.6. シャーレ底面に満遍なくセルスクレーパーをかける。
6.1.7. ピペットを用いて、シャーレから細胞懸濁液を50mL遠心管に回収する。
6.1.8. ピペットを用いて、5~10mLの培地でシャーレ底面を満遍なく洗浄し、50mL遠心管に回収する。
6.1.9. 上記6.1.8.の操作を必要回数行う。
6.1.10. 遠心分離する。
設定条件:500G 5min 4℃
6.1.11. 抗原パルスがある場合は手順「6.2.抗原パルス」、抗原パルスがない場合は手
順「6.3.細胞凍結」に進む。
DCMでパルスを実施していない細胞のみ行う。
6.2.1. 遠心分離終了後、上清を除去する。
6.2.2. タッピングでペレットを崩す。
6.2.3. ピペットを用いて、5~20mLの培地で遠心管の各細胞を1本の50mL遠心管(以下「遠心管1」)にまとめる。
※遠心機に温度調節機能がない場合、遠心管1は低接着遠心管の使用が推奨される。
6.2.4. 細胞回収後の遠心管を培地で洗いながら遠心管1にまとめ、40mL程度にメスアップする。(細胞懸濁液1)
※細胞数算定のため液量を量る。
6.2.5. 5回以上転倒混和した後、マイクロピペットを用いてマイクロチューブに50μL採取する。
6.2.6. トリパンブルー染色液で2~20倍に希釈し、血球計算盤を用いて生細胞数及び死細胞数をカウントする。
6.2.7. カウント数から総生細胞数を算出する。
6.2.8. 算出した総細胞数を基に、培地及び抗原の各使用量を算定する。
6.2.9. 抗原をパルスする際、2mg/mLとなるよう指定の溶媒で溶解する。
6.2.10. 遠心管に算定量の培地を量り取る。(使用する抗原の種類数、準備する。)
6.2.11. 各量り取った培地から5mL程度採取し、算定量の各抗原を溶解する。
6.2.12. 6.2.11をシリンジフィルターに通して6.2.10.の培地に添加する(「以下ペプチド添加培地」)。
6.2.13. 細胞懸濁液1を、使用抗原の種類数に遠心管に分注し、遠心分離する。
設定条件:500G 5min 4℃
6.2.14. 遠心分離終了後、50mL試験管に上清を回収する。
6.2.15. 回収した上清をサンプルチューブに1mL採取し、工程参考品とする。
(各遠心管から採取し、一つにまとめて良い)
6.2.16. タッピングでペレットを崩し、各ペプチド添加培地を加え懸濁する。
6.2.17. インキュベータに入れ、30~60分静置する。
設定条件:37℃
6.2.18. 遠心分離する。
設定条件:500G 5min 4℃
6.3.1. 遠心分離終了後、上清を除去する。
6.3.2. タッピングでペレットを除去する。
6.3.3. ピペットを用いて、各細胞を培地で洗いながらセルストレーナーをセットした50mL遠心管にまとめ、40mL程度にメスアップする(以下「細胞懸濁液2」)。
※細胞数算定のため液量を量る。
6.3.4. 5回以上転倒混和した後、マイクロピペットを用いてマイクロチューブに細胞懸濁液2を50μL採取する。
6.3.5. トリパンブルー染色液で2~20倍に希釈し、血球計算盤を用いて生細胞数及び死細胞数をカウントする。
6.3.6. カウント数から生細胞数を算出する。
6.3.7. 算出した総細胞数を基に、凍結本数及び培地並びに凍結培地使用量を算定する。6.3.8. 凍結保管用チューブを算定本数用意する。
6.3.9. 細胞懸濁液2を遠心分離する。
設定条件:500G 5min 4℃
6.3.10. 遠心分離終了後、50mL遠心管に上清を回収する。
6.3.11. 回収した上清を安全性試験のために分注する。(各遠心管から採取し、一つに
まとめて良い)
6.3.12. タッピングでペレットを崩す。
6.3.13. 培地で細胞を懸濁し、6.3.7.で算定した「培地使用量」に合せる
6.3.14. 一番細胞数が多いワクチンから、フローサイトメトリー解析検体と製品参考品
の検体を採取する。マイクロピペットを用いて、6.3.13.の懸濁液を0.1mLずつ2本
の凍結保管用チューブに採取し、それぞれに0.4mLの培地、0.5mLの凍結培地を加えて懸濁する。
6.3.15. 6.3.13.の懸濁液に、6.3.7.で算定した「凍結培地使用量」の凍結培地を添加し、緩やかにピペッティングして混和する。
6.3.16. 異物検査を実施する。
6.3.17. 凍結保管用チューブに1mLずつ、全量を分注する。
6.3.18. バイセルに凍結保管用チューブを入れ、-80℃設定のフリーザに静置する。
6.3.19. バイセルを-80℃設定のフリーザに静置後、3時間以上36時間以内に凍結
細胞を-135℃以下設定のフリーザ又は液体窒素保存容器に移動する。
5.試薬分注
5.1.DMSO分注
必要に応じて、DMSO分注を行う。
5.1.1. 注射針を装着したシリンジを用いてDMSOを適量サンプルチューブに量り取る。
5.1.2. 使用直前まで室温で保管する。
必要に応じて、蒸留水または生理食塩水分注を行う。
5.2.1. 蒸留水または生理食塩水を適量サンプルチューブに量り取る。
5.2.2. 使用直前まで4℃設定の保冷庫で保管する。
6.1. 未成熟樹状細胞回収
6.1.1. インキュベータからシャーレを取り出し、作業エリアに搬入する。
※ シャーレの内1枚、又は観察用シャーレを検鏡する。検鏡した観察用シャーレは破棄する。
※ シャーレは原則5枚1組として扱い、1組毎に全てのシャーレに対して下記6.1.2.~6.1.8.の操作を行う。
6.1.2. ピペットを用いて、シャーレ内の細胞を培養上清ごと50mL遠心管に回収する。
6.1.3. ピペットを用いて、5~10mLの培地でシャーレ底面を満遍なく洗浄し、50mL遠心管に回収する。
6.1.4. ピペットを用いて、シャーレに培地を1~2mL分注する。
6.1.5. シャーレ底面に満遍なくセルスクレーパーをかける。
6.1.6. ピペットを用いて、シャーレから細胞懸濁液を50 mL遠心管に回収する。
6.1.7. ピペットを用いて、5~10mLの培地でシャーレ底面を満遍なく洗浄し、50mL遠心管に回収する。
6.1.8. 上記6.1.7.の操作を必要回数行う。
6.1.9. 遠心分離する。
設定条件:500G 5min 4℃
6.1.10. 遠心分離終了後、上清を除去し、タッピングでペレットを崩す。
6.1.11. ピペットを用いて、5~20mLの培地で各遠心管の細胞を1本の50mL遠
心管(以下「遠心管1」)にまとめる。
6.1.12. 細胞回収後の各遠心管を培地で洗いながら遠心管1にまとめ、40mL程度に
メスアップする(以下「細胞懸濁液1」)。
※細胞数算定のため液量を量る。
6.2.1. 5回以上転倒混和した後、マイクロピペットを用いてマイクロチューブに細胞懸濁液1を50μL採取する。
6.2.2. トリパンブルー染色液で2~20倍に希釈し、血球計算盤を用いて生細胞数及び死細胞数をカウントする。
6.2.3. カウント数から総生細胞数を算出する。
6.2.4. 所定の試薬を使用しない場合は手順「6.3.培地調製(フィルトレーション無)」、使用する場合は手順「6.4.培地調製(フィルトレーション有)」の操作で培地調製を行い、
手順「6.5.細胞播種」の操作を行う。PGE2の場合は未滅菌のPGE2を使用する場合のみフィルトレーションを実施。
6.3.1. 算出した総生細胞数を基に、シャーレ枚数並びに培地、GM-CSF、IL-4、ピシバニール、PGE2の各使用量を算定する。
6.3.2. 遠心管に算定量の培地を量り取る。
6.3.3. マイクロピペットを用いて、算定量のGM-CSF、IL-4、ピシバニール、PGE2、を添加する(以下「調製培地」)。
6.4.1. 算出した総生細胞数を基に、シャーレ枚数並びに培地、GM-CSF、IL-4、ピシバニール、PGE2及び抗原の各使用量を算定する。
6.4.2. 抗原Aをパルスする場合、2mg/mLとなるようにDMSOで抗原Aを溶解する。
6.4.3. 抗原Bをパルスする場合、2mg/mlとなるように蒸留水または生理食塩水で抗原Bを溶解する。
6.4.4. 遠心管に培地を量り取る。
※ネオアンチゲンペプチドを2種類以上使用する場合は、医師の指示のもと適宜、別々の遠心管に培地を量り取り、6.4.5.から6.5.7.の操作を遠心管ごとに実施する。
また、ネオアンチゲンペプチドは本工程および次工程で使用する種類を考慮に入れる。
6.4.5. 量り取った培地から5mLを採取し、算定量の抗原A、抗原Bを添加する。
※2種類以上の抗原を使用する場合は、各抗原を別の培地に分注する。
※未滅菌のPGE2を使用する場合は、本工程で添加する。
6.4.6. マイクロピペットを用いて、6.4.4.の培地に算定量のGM-CSF、IL-4、ピシバニール、PGE2を添加する。
6.4.7. 6.4.5.をシリンジフィルターに通して6.4.6.の培地に添加する(以下「調製培地」)。
6.5.1. シャーレを算定枚数用意する。
6.5.2. 細胞懸濁液を遠心分離する。
設定条件:500G 5min 4℃
※医師の指示のもと適宜、ネオアンチゲンの種類に応じて細胞懸濁液を適当な本数の遠心管に分注する。
6.5.3. 遠心分離終了後、遠心管に上清を回収する。
6.5.4. 回収した上清をサンプルチューブに1mL採取し、工程参考品とする。
6.5.5. タッピングでペレットを崩し、調製培地を加え懸濁する。
6.5.6. ピペットを用いて、シャーレに6mLずつ分注する。
※ 分注の際は十分ピペッティングを行う。
※ 観察用シャーレには2mL分注する。
6.5.7. シャーレをインキュベータに入れ、一晩(12時間以上36時間未満)静置する。
設定条件:37℃ CO2濃度 5.0%
・最終製剤化工程:
・細胞密度の調製(2×107cells/mL)
細胞を培地に懸濁する。
・細胞凍結保存液との混合(1×107cells/mL)
細胞懸濁液(培地)と細胞凍結保存液を1:1で混合する。
使用時に2倍希釈するように調製濃度は濃く(2倍)で作成しておく。
0.1~1×107cells/mLの細胞を0.5~1mL/tubeで凍結保存することも可能。
・クライオチューブ等への充填(1×107cells/1mL/tube)
上記細胞懸濁液をクライオチューブに分注する。
・クライオチューブ(凍結保存細胞)および添付融解剤(生理食塩水)のアンプルへのラベリング
・製剤の保存:凍結保存細胞(≦-80℃)
生理食塩水(室温)
最終製剤(凍結保存細胞+添付融解剤)が得られる。
A:細胞凍結保存液にて細胞を懸濁する(1×107cells/mL)
B:クライオチューブに1mLずつ細胞懸濁液を分注する(1×107cells/1mL/tube)
実施例7で製造したパルス樹状細胞を用いて樹状細胞療法を行う。
1)デキストラン1mLに生理食塩水8mLを加えた溶液(解凍用液)を作製する。
2)液体窒素下で凍結したDC(1×107個/1mL)の入ったクライオバイアルを37℃に加温したヒートブロックを用いて7割程度まで解凍する。
3)解凍用液を遠心管に一部分注し、残りの解凍用液でDCの入ったクライオバイアルを共洗いしながら遠心管に移す(総量10ml)。
4)転倒混和後、カウント用サンプルとして50μL採取し、トリパンブルー染色液で2倍希釈する。血球計算盤を用いて生細胞数及び死細胞数をカウントし、総細胞数、生細胞率を算出する。
5)遠心分離を行う(500g、5min、4℃、アクセル・ブレーキ有)。遠心分離したら、上清除去後、タッピングでペレットを崩す。細胞懸濁液に生理食塩水を加えて約13mLにする。これを2回繰り返す。
6)凍結DCワクチンを生理食塩水0.6~0.8mL程度で再懸濁し、シリンジ(インスリン自己注射用、30G×10mm、0.5mL)に移す。
7)腋窩リンパ節近傍、鼡径リンパ節近傍、あるいは病変が表皮に近い場合はその近傍に、数ヶ所に分けて皮下~皮内投与する。
本実施例では、CTCを採取したのち、CTCに発現しているネオアンチゲン蛋白を、CTCのプロテオーム解析により同定する。プロテオーム解析の手法は以下のとおりである。
(1)約1000個から数万個のCTC浮遊液を1000~2000cpmで10~15分遠心し細胞ペレットを作製する。細胞ペレットは-80℃の低温槽あるいは液体窒素容器で保存する。
(2)界面活性剤、凍結融解、浸透圧ショック、のいずれかの方法でCTCのペレットからタンパク質を抽出する。
(3)抽出したタンパク質を、高速液体クロマトグラフ(HPLC)と三連四重極型質量分析計(MS/MS)を組合せた装置(液体クロマトグラフ質量分析計「Liquid Chromatograph-Mass Spectrometry : LC-MS/MS」)で分析する。これによりCTCに発現しているタンパク質を同定する。
種々の癌患者由来の末梢血単核球細胞を用いてCTCを単離した。癌患者として、肺癌(ステージ4)、大腸癌(ステージ4)、卵巣癌(ステージ4)、乳癌、膵臓癌(ステージ4)、十二指腸乳頭部癌(ステージ4)、大腸癌(ステージ4)、肝臓癌、多発肝癌、前立腺癌(ステージ4)、食道癌の各患者由来のPBMCを使用した。PBMCの採取、PBMCからの単球の単離、CTCの単離の各手法については、実施例1~3と同様にして行った。
種々の癌患者から単離したCTCを用いて、RNAシーケンス、またはエクソーム解析によってネオアンチゲンを同定した。結果を以下の表5~13に示した。RNAシーケンスについては実施例5と同様にして行った。表5~8がエクソーム解析によるネオアンチゲン同定の結果である。プロテオーム解析を加えることにより、蛋白として発現しているネオアンチゲンのみを絞り込んで同定することが可能となる。プロテオーム解析のかわりにRNAシーケンスをエクソーム解析に組み合わせることによってもネオアンチゲンの絞り込み同定は可能である。表9~12がRNAシーケンスによるネオアンチゲン同定の結果である。
がん以外の疾患への適用例を説明する。
(1)CTCの遺伝子解析によりドライバー遺伝子を含めた種々の遺伝子変異が明らかになり、分子標的薬の選択が可能となる。
(2)(1)と同様に高頻度マイクロサテライト不安定性(MSI-High)の検出も可能となり、免疫チェックポイント阻害薬の適応であるかを同定できる。
(3)トランスクリプトーム解析やシングルセルRNA-seqと臨床データを合わせることにより、転移を起こしている(あるいは起こしやすい)症例と起こしていない(あるいは起こしにくい)症例でのCTCの差異を明らかにすることができる。これにより症例の予後予測や転移の生じるメカニズムの解明につながる。
(4)メタボローム解析(メタボローム解析は対象とするサンプルに存在する代謝産物の全ての種類や濃度を網羅的に検出し、その結果を解析することを指し、メタボロミクスとも呼称される)を行うことにより、CTCの代謝特性が明らかなり、(3)の検討がより包括的に可能となる。
健康状態を確認する場合の適用例を説明する。
(1)CTCのエクソーム解析により、悪性腫瘍がレアバリアント変異によるかが検出できる。さらに、血縁者の検査を行うことにより同じ疾患が存在することが明らかになる場合があり、血縁者の早期診断・早期治療が可能となる。
(2)ネオアンチゲン同定の際にはCTCに加え、正常細胞のDNAも抽出できる。この正常DNAの全ゲノム解析あるいはDNAチップ解析により、正常の生殖細胞系列のゲノム配列が決定できる。この配列を元に疾患に関わるレアバリアント変異だけでなくコモンバリアントのリスク計算が可能となる。悪性腫瘍以外の疾患に対する予防医療にも役立つ。
以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願及び他の文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。本願は、日本国特許庁に2021年6月30日に出願された特願2021-108784に対して優先権主張をするものであり、その内容はその全体があたかも本願の内容を構成するのと同様に参考として援用される。
配列番号23~98:実施例11で同定したネオアンチゲン
Claims (39)
- 核酸増幅及び/または培養による増殖をせずに解析するための被験者由来の血中循環腫瘍細胞を含む試料を調製する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
必要に応じて、該解析に必要な血中循環腫瘍細胞の純度を確認する工程と、
必要に応じて、該血中循環腫瘍細胞に対して、該解析のための前処理を行う工程と
を含む、方法。 - 前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、請求項1に記載の方法。
- 前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、請求項1または2に記載の方法。
- 前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、請求項1~3のいずれか一項に記載の方法。
- 前記血中循環腫瘍細胞が約1×103個以上である、請求項1~4のいずれか一項に記載の方法。
- 前記末梢血単核球細胞が約1×104個以上である、請求項1~5のいずれか一項に記載の方法。
- 前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、請求項4に記載の方法。
- さらに、前記血中循環腫瘍細胞を凍結する工程を含む、請求項1~7のいずれか一項に記載の方法。
- 前記解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、請求項1~8のいずれか一項に記載の方法。
- 前記試料が、前記解析を行うことによって前記被験者が有するネオアンチゲンを同定するためのものである、請求項1~9のいずれか一項に記載の方法。
- さらに、前記血中循環腫瘍細胞からDNA、RNA、またはタンパク質を抽出する工程を含む、請求項1~10のいずれか一項に記載の方法。
- 前記DNAまたはRNAが、少なくとも約100pg抽出される、請求項11に記載の方法。
- 核酸増幅及び/または培養による増殖をせずに被験者由来の血中循環腫瘍細胞を解析する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞を用いて解析を行う工程と
を含む、方法。 - 前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、請求項13に記載の方法。
- 前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、請求項13または14に記載の方法。
- 前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、請求項13~15のいずれか一項に記載の方法。
- 前記血中循環腫瘍細胞が約1×103個以上である、請求項13~16のいずれか一項に記載の方法。
- 前記末梢血単核球細胞が約1×104個以上である、請求項13~17のいずれか一項に記載の方法。
- 前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、請求項16に記載の方法。
- さらに、前記血中循環腫瘍細胞を凍結する工程を含む、請求項13~19のいずれか一項に記載の方法。
- 該解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、請求項13~20のいずれか一項に記載の方法。
- 請求項13~21のいずれか一項に記載の方法によって前記被験者由来の前記血中循環腫瘍細胞を解析することにより、前記被験者が有するネオアンチゲンを同定する方法。
- さらに、前記血中循環腫瘍細胞からDNA、RNA、またはタンパク質を抽出する工程を含む、請求項22に記載の方法。
- さらに、前記DNAまたはRNAからDNA/RNAシーケンスライブラリを作成する工程と、
前記シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と
を含み、前記血中循環腫瘍細胞の純度が少なくとも約20%である、請求項24に記載の方法。 - 前記解析が、前記DNAの全エクソーム解析を含む、請求項24に記載の方法。
- 前記解析が、さらに、前記血中循環腫瘍細胞のプロテオーム解析を含む、請求項25に記載の方法。
- 前記DNAまたはRNAが、少なくとも約100pg抽出される、請求項23~26のいずれか一項に記載の方法。
- 被験者が有するネオアンチゲンを用いた樹状細胞療法のための細胞を製造する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞からDNAまたはRNAを抽出し、DNA/RNAシーケンスライブラリを作成する工程と、
該シーケンスライブラリの変異情報に基づき、ネオアンチゲンを同定する工程と、
該ネオアンチゲンを樹状細胞に導入する工程と
を含む、方法。 - 前記アフェレーシス条件が、スピルオーバーボリューム及び/またはバフィーコートボリュームを血中循環腫瘍細胞濃縮条件にすることを含む、請求項28に記載の方法。
- 前記アフェレーシス条件が、約9~約12mlのスピルオーバーボリューム、及び/または約7~約10mlのバフィーコートボリュームを含む、請求項28または29に記載の方法。
- 前記血中循環腫瘍細胞を単離する工程は、モノクローナル抗体を用いて、またはサイズ分画によって行われる、請求項28~30のいずれか一項に記載の方法。
- 前記血中循環腫瘍細胞が約1×103個以上である、請求項28~31のいずれか一項に記載の方法。
- 前記末梢血単核球細胞が約1×104個以上である、請求項28~32のいずれか一項に記載の方法。
- 前記モノクローナル抗体が、抗EpCAM抗体、抗Vimentin抗体、抗N-Cadherin抗体、抗CD20抗体、抗E-Cadherin抗体、抗Desmoglein-3抗体、抗Syndecan-1抗体、抗CD99抗体、抗CD81抗体、及びPAX3抗体を含む、請求項31に記載の方法。
- さらに、前記血中循環腫瘍細胞を凍結する工程を含む、請求項28~34のいずれか一項に記載の方法。
- 前記DNAまたはRNAが、少なくとも約100pg抽出される、請求項28~35のいずれか一項に記載の方法。
- 前記樹状細胞は、前記アフェレーシスによって前記被験者から得られたものであるか、または前記単球から誘導されるものである、請求項28~36のいずれか一項に記載の方法。
- 核酸増幅及び/または培養による増殖をせずに血中循環腫瘍細胞を解析することにより被験者を治療または予防する方法であって、
アフェレーシスを用いて該被験者から採取された末梢血単核球細胞から単球を単離する工程であって、前記アフェレーシスを行うアフェレーシス条件が、血中循環腫瘍細胞濃縮条件を含む、工程と、
単球を単離した該末梢血単核球細胞から、血中循環腫瘍細胞を単離する工程と、
該血中循環腫瘍細胞を用いて解析を行う工程と、
該解析結果に基づいて、該被験者を治療または予防する工程と
を含む、方法。 - 該解析が、全エクソーム解析、全ゲノム解析、RNA-Seq、シングルセルRNA-Seq、プロテオーム解析、およびトランスクリプトーム解析を含む、請求項38に記載の方法。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202280047045.7A CN117881794A (zh) | 2021-06-30 | 2022-06-29 | 制备包含血中循环肿瘤细胞的试样的方法 |
JP2023515199A JP7426165B2 (ja) | 2021-06-30 | 2022-06-29 | 血中循環腫瘍細胞を含む試料を調製する方法 |
EP22833232.6A EP4365204A1 (en) | 2021-06-30 | 2022-06-29 | Method for preparing sample containing tumor cells circulating in blood |
JP2023187279A JP2024016131A (ja) | 2021-06-30 | 2023-10-31 | 血中循環腫瘍細胞を含む試料を調製する方法 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2021108784 | 2021-06-30 | ||
JP2021-108784 | 2021-06-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023277086A1 true WO2023277086A1 (ja) | 2023-01-05 |
Family
ID=84691857
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2022/026036 WO2023277086A1 (ja) | 2021-06-30 | 2022-06-29 | 血中循環腫瘍細胞を含む試料を調製する方法 |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP4365204A1 (ja) |
JP (2) | JP7426165B2 (ja) |
CN (1) | CN117881794A (ja) |
TW (1) | TW202317754A (ja) |
WO (1) | WO2023277086A1 (ja) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140154799A1 (en) * | 2012-12-05 | 2014-06-05 | The Royal Institution For The Advancement Of Learning/Mcgill University | Method of obtaining circulating cancer cell populations, associated cellular compositions and methods of using same |
JP2018512380A (ja) * | 2015-03-13 | 2018-05-17 | サイズ セル セラピー コーポレイション | 活性化t細胞を用いるがん治療の方法 |
JP2018527935A (ja) * | 2015-09-10 | 2018-09-27 | キャンサー・リサーチ・テクノロジー・リミテッド | 癌における「免疫チェックポイント介入」 |
JP2021108784A (ja) | 2020-01-07 | 2021-08-02 | キヤノンメディカルシステムズ株式会社 | X線診断装置 |
-
2022
- 2022-06-29 JP JP2023515199A patent/JP7426165B2/ja active Active
- 2022-06-29 TW TW111124370A patent/TW202317754A/zh unknown
- 2022-06-29 CN CN202280047045.7A patent/CN117881794A/zh active Pending
- 2022-06-29 EP EP22833232.6A patent/EP4365204A1/en active Pending
- 2022-06-29 WO PCT/JP2022/026036 patent/WO2023277086A1/ja active Application Filing
-
2023
- 2023-10-31 JP JP2023187279A patent/JP2024016131A/ja active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140154799A1 (en) * | 2012-12-05 | 2014-06-05 | The Royal Institution For The Advancement Of Learning/Mcgill University | Method of obtaining circulating cancer cell populations, associated cellular compositions and methods of using same |
JP2018512380A (ja) * | 2015-03-13 | 2018-05-17 | サイズ セル セラピー コーポレイション | 活性化t細胞を用いるがん治療の方法 |
JP2018527935A (ja) * | 2015-09-10 | 2018-09-27 | キャンサー・リサーチ・テクノロジー・リミテッド | 癌における「免疫チェックポイント介入」 |
JP2021108784A (ja) | 2020-01-07 | 2021-08-02 | キヤノンメディカルシステムズ株式会社 | X線診断装置 |
Non-Patent Citations (2)
Title |
---|
JUNICHI TAGUCHI: "Neoantigen dendritic cell vaccine therapy", EXPLANATORY DOCUMENT, 1ST DOCUMENT, MEDICAL CORPORATION MIDTOWN CLINIC TOKYO MIDTOWN CLINIC, JP, JP, pages 1 - 12, XP009542580, Retrieved from the Internet <URL:https://saiseiiryo.mhlw.go.jp/published_plan/index/1/3> [retrieved on 20220913] * |
LI HEMING; MENG QING H.; NOH HYANGSOON; BATTH IZHAR SINGH; SOMAIAH NEETA; TORRES KEILA E.; XIA XUEQING; WANG RUOYU; LI SHULIN: "Detection of circulating tumor cells from cryopreserved human sarcoma peripheral blood mononuclear cells", CANCER LETTERS, NEW YORK, NY, US, vol. 403, 23 June 2017 (2017-06-23), US , pages 216 - 223, XP085144906, ISSN: 0304-3835, DOI: 10.1016/j.canlet.2017.05.032 * |
Also Published As
Publication number | Publication date |
---|---|
CN117881794A (zh) | 2024-04-12 |
JPWO2023277086A1 (ja) | 2023-01-05 |
EP4365204A1 (en) | 2024-05-08 |
JP2024016131A (ja) | 2024-02-06 |
TW202317754A (zh) | 2023-05-01 |
JP7426165B2 (ja) | 2024-02-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6761850B2 (ja) | 濾過を通して生物学的サンプルから抽出された又は単離された希少細胞の多重分析のための方法 | |
Aliotta et al. | Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription | |
JP2008529541A (ja) | 腫瘍細胞の分離用容器 | |
US20200368336A1 (en) | Method for preparing personalized cancer vaccine | |
US20180031543A1 (en) | Devices, solutions and methods for sample collection related applications, analysis and diagnosis | |
TW201130978A (en) | Method of isolation of stem cell populations from peripheral blood using sized-based separation (elutriation) | |
Steininger et al. | First comparative evaluation of a new leukapheresis technology in non‐cytokine‐stimulated donors | |
JP5295950B2 (ja) | 移植片対宿主病または移植関連死に対する感受性を検出するための方法および試薬 | |
Guldner et al. | Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes | |
WO2023277086A1 (ja) | 血中循環腫瘍細胞を含む試料を調製する方法 | |
Tan et al. | Presence of tumor cells in intra-operative blood salvage autotransfusion samples from hepatocellular carcinoma liver transplantation: analysis using highly sensitive microfluidics technology | |
KR20220116475A (ko) | 시퀀싱을 위한 핵산을 획득하기 위한 방법 | |
Strasser et al. | Comparison of two apheresis systems for the collection of CD14+ cells intended to be used in dendritic cell culture | |
Wu et al. | Decision‐tree algorithm for optimized hematopoietic progenitor cell–based predictions in peripheral blood stem cell mobilization | |
Strasser et al. | Comparison of two mononuclear cell program settings on two apheresis devices intended to collect high yields of CD14+ and CD3+ cells | |
Mangiola et al. | Transcriptome sequencing and multi-plex imaging of prostate cancer microenvironment reveals a dominant role for monocytic cells in progression | |
RU2554746C1 (ru) | Способ получения суммарной фракции внеклеточных нуклеиновых кислот из крови | |
Debuque et al. | Methods for axolotl blood collection, intravenous injection, and efficient leukocyte isolation from peripheral blood and the regenerating limb | |
Seghatchian et al. | Unresolved clinical aspects and safety hazards of blood derived-EV/MV in stored blood components: from personal memory lanes to newer perspectives on the roles of EV/MV in various biological phenomena | |
Ciccocioppo et al. | Reduced number and function of peripheral dendritic cells in coeliac disease | |
Schedel et al. | Genome-wide platelet RNA profiling in clinical samples | |
Stoecklein | CTC-Based Liquid Biopsies and Diagnostic Leukapheresis | |
Putz et al. | Generation of clinical-grade monocyte-derived dendritic cells using the CliniMACS system | |
RU2706714C1 (ru) | Способ ранней молекулярно-биологической диагностики злокачественных новообразований и бокового амиотрофического склероза | |
Dannull et al. | Transfecting Human Monocytes with RNA |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22833232 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2023515199 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 202280047045.7 Country of ref document: CN |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2022833232 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2022833232 Country of ref document: EP Effective date: 20240130 |