CN115948341A - CAR-immunocyte for knocking down NKG2A gene and application thereof - Google Patents
CAR-immunocyte for knocking down NKG2A gene and application thereof Download PDFInfo
- Publication number
- CN115948341A CN115948341A CN202211504757.5A CN202211504757A CN115948341A CN 115948341 A CN115948341 A CN 115948341A CN 202211504757 A CN202211504757 A CN 202211504757A CN 115948341 A CN115948341 A CN 115948341A
- Authority
- CN
- China
- Prior art keywords
- car
- cells
- immune cell
- tumor
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 101150110881 NKG2A gene Proteins 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 218
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 57
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 15
- 230000007246 mechanism Effects 0.000 claims abstract description 10
- 230000017188 evasion or tolerance of host immune response Effects 0.000 claims abstract description 9
- 201000011510 cancer Diseases 0.000 claims abstract description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract 2
- 210000000822 natural killer cell Anatomy 0.000 claims description 23
- 108091033409 CRISPR Proteins 0.000 claims description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 15
- 239000008194 pharmaceutical composition Substances 0.000 claims description 14
- 230000003834 intracellular effect Effects 0.000 claims description 13
- 238000010354 CRISPR gene editing Methods 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 9
- 210000002865 immune cell Anatomy 0.000 claims description 9
- 230000004068 intracellular signaling Effects 0.000 claims description 7
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 6
- 108091007433 antigens Proteins 0.000 claims description 6
- 102000036639 antigens Human genes 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 claims description 4
- 210000000581 natural killer T-cell Anatomy 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 230000000139 costimulatory effect Effects 0.000 claims description 3
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 claims description 2
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 claims description 2
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 claims description 2
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 2
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 2
- -1 CLDN 18.2 Proteins 0.000 claims description 2
- 101150084967 EPCAM gene Proteins 0.000 claims description 2
- 102100032530 Glypican-3 Human genes 0.000 claims description 2
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 2
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 2
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 claims description 2
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 claims description 2
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 claims description 2
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 2
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 claims description 2
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 claims description 2
- 101001136592 Homo sapiens Prostate stem cell antigen Proteins 0.000 claims description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 2
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 2
- 102100034256 Mucin-1 Human genes 0.000 claims description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 2
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 claims description 2
- 102100040120 Prominin-1 Human genes 0.000 claims description 2
- 102100036735 Prostate stem cell antigen Human genes 0.000 claims description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 2
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 2
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 2
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 claims description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 claims 2
- 102100025096 Mesothelin Human genes 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 101150069255 KLRC1 gene Proteins 0.000 abstract description 55
- 101100404845 Macaca mulatta NKG2A gene Proteins 0.000 abstract description 55
- 102100022682 NKG2-A/NKG2-B type II integral membrane protein Human genes 0.000 abstract description 55
- 102100028970 HLA class I histocompatibility antigen, alpha chain E Human genes 0.000 abstract description 21
- 101000986085 Homo sapiens HLA class I histocompatibility antigen, alpha chain E Proteins 0.000 abstract description 21
- 210000004881 tumor cell Anatomy 0.000 abstract description 12
- 102100037850 Interferon gamma Human genes 0.000 abstract description 10
- 108010074328 Interferon-gamma Proteins 0.000 abstract description 10
- 230000028327 secretion Effects 0.000 abstract description 10
- 239000003446 ligand Substances 0.000 abstract description 8
- 230000004083 survival effect Effects 0.000 abstract description 8
- 230000030279 gene silencing Effects 0.000 abstract description 7
- 230000002401 inhibitory effect Effects 0.000 abstract description 6
- 108020004414 DNA Proteins 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 4
- 230000005909 tumor killing Effects 0.000 abstract description 4
- 102000053602 DNA Human genes 0.000 abstract 1
- 238000012226 gene silencing method Methods 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 31
- 102000003735 Mesothelin Human genes 0.000 description 16
- 108090000015 Mesothelin Proteins 0.000 description 16
- 206010033128 Ovarian cancer Diseases 0.000 description 15
- 206010061535 Ovarian neoplasm Diseases 0.000 description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 201000010099 disease Diseases 0.000 description 13
- 241000700605 Viruses Species 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 230000002147 killing effect Effects 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 8
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 8
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 8
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 8
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000004806 packaging method and process Methods 0.000 description 8
- 241000713666 Lentivirus Species 0.000 description 7
- 108091008042 inhibitory receptors Proteins 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 102000001398 Granzyme Human genes 0.000 description 5
- 108060005986 Granzyme Proteins 0.000 description 5
- 102000004503 Perforin Human genes 0.000 description 5
- 108010056995 Perforin Proteins 0.000 description 5
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 229930192851 perforin Natural products 0.000 description 5
- 206010029260 Neuroblastoma Diseases 0.000 description 4
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 4
- 230000003698 anagen phase Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000003197 gene knockdown Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 3
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 3
- 201000002528 pancreatic cancer Diseases 0.000 description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 206010050685 Cytokine storm Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 201000010915 Glioblastoma multiforme Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 102100028113 Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Human genes 0.000 description 2
- 101000916625 Homo sapiens Granulocyte-macrophage colony-stimulating factor receptor subunit alpha Proteins 0.000 description 2
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 2
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 2
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010033701 Papillary thyroid cancer Diseases 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 102000007327 Protamines Human genes 0.000 description 2
- 108010007568 Protamines Proteins 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 208000006990 cholangiocarcinoma Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 229920006176 crosslinked polyethyleneglycol Polymers 0.000 description 2
- 206010052015 cytokine release syndrome Diseases 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 201000010536 head and neck cancer Diseases 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 2
- 239000012642 immune effector Substances 0.000 description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 239000003978 infusion fluid Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 208000021601 lentivirus infection Diseases 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 201000002120 neuroendocrine carcinoma Diseases 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 201000002740 oral squamous cell carcinoma Diseases 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 229940048914 protamine Drugs 0.000 description 2
- 206010038038 rectal cancer Diseases 0.000 description 2
- 201000001275 rectum cancer Diseases 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 208000030045 thyroid gland papillary carcinoma Diseases 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000589301 Homo sapiens Natural cytotoxicity triggering receptor 1 Proteins 0.000 description 1
- 101000589305 Homo sapiens Natural cytotoxicity triggering receptor 2 Proteins 0.000 description 1
- 101000971513 Homo sapiens Natural killer cells antigen CD94 Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 102100021462 Natural killer cells antigen CD94 Human genes 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 238000000516 activation analysis Methods 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 229940126546 immune checkpoint molecule Drugs 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000008297 liquid dosage form Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000008299 semisolid dosage form Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 239000012439 solid excipient Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a CAR-immune cell. The NKG2A gene of the CAR-immune cell is silenced, the NKG2A gene silencing is achieved by knocking out the NKG2A gene of the CAR-immune cell by a dna molecule comprising the sequence as set forth in SEQ ID NO: 8-10, at least one sgRNA in the nucleotide sequences shown in the specification. The CAR-immune cell can block or cancel the combination of NKG2A and ligand HLA-E, and prevent the CAR-immune cell entering a tumor microenvironment from being inhibited by an inhibitory microenvironment, and the CAR-immune cell has stronger tumor killing activity and IFN-gamma secretion capacity, can effectively inhibit the growth of the tumor cell and obviously prolong the survival period of a mouse, and can be used for eliminating or relieving an immune escape mechanism of the tumor or preventing and/or treating the tumor or cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a CAR-immune cell for knocking down NKG2A gene and application thereof, and more particularly relates to a CAR-immune cell, a pharmaceutical composition and application thereof.
Background
In recent years, chimeric antigen receptor T (CAR-T) cells have shown remarkable effects in the treatment of hematological malignancies. However, the CAR-T cells are easy to generate adverse reactions such as cytokine storm, neurotoxicity, GVHD and the like in clinical application, and the CAR-T cells have unsatisfactory treatment effect on solid tumors, so that the clinical application of the CAR-T cells still faces challenges.
The CAR-NK cells have the advantage of good safety compared with CAR-T cells, and generally do not cause side effects such as cytokine storm and GVHD; the NK cells can play a role in directly killing tumor cells without antigen presentation and MHC restriction; the CAR-NK cell can recognize and kill tumors by various recognition mechanisms such as CAR dependence and NKR dependence, and has a wide anti-tumor spectrum. Therefore, the CAR-NK cells have wide application prospects in antitumor therapy, and become hot spots in the research and development field of cellular immunotherapy. However, both CAR-NK cells and CAR-T cells face the challenge of being susceptible to the tumor immunosuppressive microenvironment.
Thus, there is a continuing development of a CAR-immune cell that is not susceptible to tumor immunosuppressive microenvironments.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the CAR-immune cell for knocking down the NKG2A gene has the advantages of resisting the down-regulation effect of a tumor immune microenvironment, resisting cell depletion and the like.
The present invention has been completed based on the following findings of the inventors:
at present, the method for reversing the tumor microenvironment to cause the immune cell exhaustion usually adopts an immune checkpoint blocking therapy, namely, the method blocks the binding of inhibitory receptors on the surfaces of T cells or NK cells and corresponding ligands on the surfaces of tumor cells or regulatory T cells, myeloid cells or stromal cells in the tumor microenvironment by using an immune checkpoint inhibitor, such as monoclonal antibodies of immune checkpoint molecules, so as to weaken the function inhibition of immune effector cells such as T cells or NK cells and arouse the capability of the immune effector cells to effectively kill tumors. However, clinically, the response rate of immune checkpoint blockade therapy is low, only 20% to 30%, and drug resistance is easily generated.
NKG2A acts as an important immune checkpoint and is expressed primarily on the NK cell surface, as well as on NKT cells and certain CD8+ T cell subsets. NKG2A and CD94 are expressed on the cell surface in the form of heterodimer, mainly recognize non-classical MHC-I molecule HLA-E, transmit inhibitory signals to NK cells, and inhibit the effect function of the NK cells, namely, the NKG2A highly expressed in the tumor microenvironment can weaken the activation and anti-tumor functions of immune cells such as the NK cells.
Based on the above, the inventor finds that compared with an immune checkpoint inhibitor, the CAR-immune cell with the knocked-down NKG2A gene does not need to prepare and develop a monoclonal antibody, and the knocked-down NKG2A expression can directly block or cancel the combination of the NKG2A gene and ligand HLA-E, so that the induction of inhibitory signals from a tumor microenvironment is blocked, the CAR-immune cell entering the tumor microenvironment is prevented from being inhibited by the inhibitory microenvironment, the exhaustion is effectively resisted, and the CAR-immune cell can fully play an anti-tumor role.
Thus, in one aspect of the invention, the invention features a CAR-immune cell. According to embodiments of the invention, the NKG2A gene of the CAR-immune cell is silenced; wherein the immune cells of the CAR-immune cells comprise at least one of NK cells, T cells, NKT cells, and γ δ T cells. The CAR-immune cells (especially CAR-NK cells) according to the embodiments of the present invention can block or abolish the binding of NKG2A to ligand HLA-E, prevent the CAR-immune cells entering the tumor microenvironment from being inhibited by the inhibitory microenvironment, and the CAR-immune cells have stronger tumor killing activity and IFN-gamma secretion ability, can effectively inhibit the growth of tumor cells and prolong the survival of mice, can be used for eliminating or alleviating the immune escape mechanism of tumors or for preventing and/or treating tumors or cancers.
According to an embodiment of the invention, the immune cell of the CAR-immune cell is an NK cell.
It should be noted that, in the present invention, the source of the NK cells of the CAR-NK cells is not particularly limited, and includes but is not limited to NK cells of different sources such as NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, iPSC-derived NK cells, NK-92 cells, and the like, and is also applicable to CAR-NK cells, CAR-T cells, CAR-NKT cells, and CAR- γ δ T cells.
According to an embodiment of the invention, said silencing of the NKG2A gene is achieved by knocking out the NKG2A gene of said CAR-immune cell.
According to an embodiment of the invention, the knockout is achieved by the CRISPR/Cas9 system.
According to embodiments of the invention, the sgRNA of the CRISPR/Cas9 system has the sequence as set forth in SEQ ID NO:8 to 10 in sequence. Therefore, the sgRNA can be used for targeted knock-down of NKG2A gene in CAR-immune cells (especially CAR-NK cells) to obtain the CAR-immune cells capable of silencing the NKG2A gene, and the application of the sgRNA can further reduce the expression of other inhibitory receptors such as CAR-immune cells PD-1 and Tim-3, so that the tumor killing activity and IFN-gamma secretion capacity of the CAR-immune cells are improved, the growth of the tumor cells is effectively inhibited, and the survival period of mice is prolonged.
According to an embodiment of the invention, the knockout is performed by: and introducing an expression vector carrying the sgRNA and the nucleic acid encoding the Cas9 molecule into the CAR-immune cell to be modified for culture treatment.
According to the embodiment of the invention, the time of the culture treatment is 24-72 h.
According to an embodiment of the invention, the expression vector is a eukaryotic cell expression vector.
According to an embodiment of the invention, the CAR of the CAR-immune cell comprises: an extracellular region, wherein the extracellular region comprises a single-chain antibody and a hinge region, the C end of the single-chain antibody is connected with the N end of the hinge region, and the single-chain antibody specifically recognizes a tumor antigen MSLN; a transmembrane region, wherein the N end of the transmembrane region is connected with the C end of the hinge region of the extracellular region; an intracellular domain, wherein the N-terminus of said intracellular domain is linked to the C-terminus of said transmembrane domain.
According to an embodiment of the invention, the antigen comprises at least one selected from the group consisting of Mesothelin (MSLN), HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, epCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, BCMA; preferably, the antigen is MSLN.
According to an embodiment of the present invention, the single chain antibody comprises at least one selected from the group consisting of an anti-Mesothelin (MSLN) single chain antibody, an anti-HER 2 single chain antibody, an anti-EGFR single chain antibody, an anti-GPC 3 single chain antibody, an anti-MUC 1 single chain antibody, an anti-CEA single chain antibody, an anti-CLDN 18.2 single chain antibody, an anti-EpCAM single chain antibody, an anti-GD 2 single chain antibody, an anti-PSCA single chain antibody, an anti-CD 133 single chain antibody, an anti-CD 19 single chain antibody, an anti-CD 20 single chain antibody, an anti-CD 22 single chain antibody, an anti-CD 30 single chain antibody, an anti-CD 33 single chain antibody, and an anti-BCMA single chain antibody; preferably an anti-MSLN single chain antibody.
According to an embodiment of the present invention, the single chain antibody has an amino acid sequence shown as SEQ ID NO. 11. Thus, the single-chain antibody of the CAR-immune cell can be targeted to bind to mesothelin, thereby targeted killing mesothelin-positive tumor cells (e.g., pancreatic cancer, ovarian cancer, mesothelioma, etc.).
According to an embodiment of the invention, said hinge region is selected from the hinge region of the CD8 molecule.
According to an embodiment of the invention, the hinge region has the amino acid sequence shown as SEQ ID NO 12.
According to an embodiment of the invention, said transmembrane region is selected from a transmembrane segment of the CD8 molecule.
According to an embodiment of the present invention, the transmembrane region has the amino acid sequence shown as SEQ ID NO 13.
According to an embodiment of the present invention, the intracellular region comprises a co-stimulatory domain and an intracellular signaling domain.
According to an embodiment of the invention, the co-stimulatory domain is selected from the intracellular segment of the 41BB molecule.
According to an embodiment of the invention, the co-stimulatory domain has the amino acid sequence as shown in SEQ ID NO 14.
According to an embodiment of the invention, the intracellular signaling domain is selected from the intracellular segment of the CD3 ζ molecule.
According to an embodiment of the present invention, the intracellular signaling domain has an amino acid sequence as set forth in SEQ ID NO. 15.
In another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises: the aforementioned CAR-immune cell. The pharmaceutical composition according to the embodiment of the present invention can be used for eliminating or reducing immune escape mechanism of tumor or for preventing and/or treating tumor or cancer.
According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
In a further aspect of the invention, the invention proposes the use of a CAR-immune cell as described above or a pharmaceutical composition as described above for the preparation of a medicament for eliminating or reducing the immune escape mechanism of a tumor.
In a further aspect of the invention, the invention proposes the use of a CAR-immune cell as described above or a pharmaceutical composition as described above for the preparation of a medicament for the prevention and/or treatment of a tumor or cancer.
As used herein, the term "cancer" or "tumor" can be any unregulated cell growth. Illustratively, there may be non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myelogenous leukemia, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, and the like.
According to an embodiment of the invention, the tumor is a solid tumor or a hematological tumor. Including but not limited to non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, large cell neuroendocrine cancer, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer, leukemia, lymphoma.
In yet another aspect of the invention, the invention features a method of eliminating or reducing immune escape mechanisms of a tumor or preventing and/or treating a tumor or cancer. According to an embodiment of the invention, the method comprises: administering to the subject a pharmaceutically acceptable amount of the foregoing CAR-immune cell. As described above, according to the aforementioned CAR-immune cells (e.g., CAR-NK cells), NKG2A of the immune cells (e.g., NK cells) can be directly blocked or abolished from binding to ligand HLA-E, and immune escape mechanism of tumor can be eliminated or reduced, and tumor cell growth can be effectively inhibited or killed, thereby being useful for preventing and/or treating tumor or cancer. In addition, the pharmaceutical composition containing the CAR-immune cell can effectively eliminate or reduce the immune escape mechanism of the tumor, and inhibit the growth of the tumor cell or kill the tumor cell. Thus, the method can eliminate or reduce immune escape mechanisms of tumors or be used for the prevention and/or treatment of tumors or cancers.
The effective amount of the CAR-immune cells and pharmaceutical compositions of the invention may vary with the mode of administration and the severity of the disease to be treated, among other things. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as urgently required by the condition being treated.
The CAR-immune cells and pharmaceutical compositions of the invention can be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions.
In yet another aspect of the invention, the invention features a method of reducing PD-1 and/or Tim-3 expression of a CAR-immune cell. According to an embodiment of the invention, the method comprises: the peptide as shown in SEQ ID NO: 8-10 of the sgRNA molecules shown in the nucleotide sequence. Introduction of sgrnas into cells according to the methods of the embodiments of the invention can reduce expression of not only CAR-immune cells (particularly CAR-NK cells) NKG2A, but can further reduce expression of CAR-immune cells PD-1 and/or Tim-3. For example, in scientific research, the method is used for reducing the expression of various inhibitory receptors such as NKG2A, PD-1 and/or Tim-3 of cells and obtaining cells meeting the target for subsequent research.
According to an embodiment of the invention, the immune cells of the CAR-immune cells comprise at least one of NK cells, T cells, NKT cells and γ δ T cells. Illustratively, the immune cell is an NK cell or a T cell.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a CRISPR NKG2A KO plasmid map in example 1 of the present invention;
fig. 2 shows editing efficiency of different sgRNA sequences in example 1 of the present invention;
FIG. 3 shows NK-92 and MSLN-CAR-NKG2A in example 2 of the present invention low The expression level of NKG2A in NK-92 cells;
FIG. 4 shows NK-92, alpha-MSLN-CAR-NK-92, and alpha-MSLN-CAR-NKG 2A according to example 2 low Expression levels of PD-1 and Tim-3 in NK-92 cells;
FIG. 5 shows the expression of Mesothelin (MSLN) on the cell surfaces of A1847, hey, HO8910 and A2780 in example 2 of the present invention;
FIG. 6 shows the expression of HLA-E on the cell surface of A1847, HO8910 and A2780 in example 2;
FIG. 7 shows NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-NKG 2A in example 2 of the present invention low Killing efficiency of NK-92 cells to different target cells;
FIG. 8 shows NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-NKG 2A in example 2 of the present invention low Levels of granzyme B and perforin secretion by NK-92 cells;
FIG. 9 shows NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-NKG 2A in example 2 of the present invention low The IFN-gamma secretory capacity of NK-92 cells;
FIG. 10 is a flow chart of subcutaneous tumor loading of ovarian cancer A1847 cells in accordance with the invention of example 3;
FIG. 11 shows NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-NKG 2A in example 3 of the present invention low Tumor volume in NK-92 treated mice;
FIG. 12 shows NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-NKG 2A in example 3 of the present invention low Survival of mice in the NK-92 treated group.
Detailed Description
The following describes in detail embodiments of the present invention. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
In this document, the terms "comprise" or "comprise" are open-ended expressions that include the elements indicated in the present invention, but do not exclude other elements.
As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.
The term "pharmaceutical composition" as used herein generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.
As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except insofar as any conventional adjuvant is incompatible with the compounds of the invention, e.g., any adverse biological effect produced or interaction in a deleterious manner with any other component of a pharmaceutically acceptable composition, their use is contemplated by the present invention.
In this context, the term "treatment" is intended to mean the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including but not limited to the administration of a drug containing a compound described herein to an individual in need thereof.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.
Example 1: NKG2A low Preparation of CAR-NK cells
Construction of CAR expression plasmid and CRISPR NKG2A KO plasmid
1.1 construction of CRISPR NKG2A KO plasmid
According to the method for knocking down NKG2A, firstly, a sgRNA sequence for effectively silencing NKG2A is screened by adopting a gene mutation detection method, and then a CRISPR/Cas9 vector (LentiCRISPR-NKG 2A-KO) for silencing NKG2A is constructed. The CRISPR NKG2A KO plasmid map is shown in fig. 1.
Wherein, the nucleotide sequence of sgRNA1 is shown as SEQ ID NO:8 is shown in the specification; the nucleotide sequence of sgRNA2 is shown as SEQ ID NO:9 is shown in the figure; the nucleotide sequence of sgRNA3 is shown as SEQ ID NO: shown at 10.
GAAGCTCATTGTTGGGATCC(SEQ ID NO：8)；
AACAACTATCGTTACCACAG(SEQ ID NO：9)；
TGAACAGGAAATAACCTATG(SEQ ID NO：10)。
Designing sgRNA of targeted NKG2A through synthego website, annealing and phosphorylating the sgRNA, carrying out enzyme digestion on a Lent CRISPR V2 vector by using Esp3I restriction endonuclease, then connecting the phosphorylated NKG2A-sgRNA with a linearized vector, carrying out shake bacteria experiment by selecting a monoclonal colony through transformation and plate coating, sending bacterial liquid to a sequencing company for sequencing, and proving that the sequence of the inserted gene fragment is correct, wherein the result shows that the LentiCRISPR-NKG2A-KO vector is successfully constructed.
Screening of NKG2A-sgRNA sequence: in order to select sgRNA sequences with the highest NKG2A silencing efficiency, lentiCRISPR-NKG2A-KO plasmids are transfected into 293T cells, the cells are collected after 12h liquid change and 48h, and genomes are extracted. After the PCR product obtained by amplifying a specific region containing the sgRNA targeting site by using a conventional PCR technology is annealed, because a mutated single chain and an unformed wild-type chain cannot be completely complementary and paired, a bulge is generated at the mutated position, and then the bulge is recognized and cut by T7E1 to form two broken short chains. The bands obtained from the agarose gel electrophoresis experiments were subjected to grayscale analysis using Imagine J, the results of which are shown in figure 2. The result shows that the editing efficiency of sgRNA2 is higher compared with sgRNA1 and sgRNA3, and a subsequent experiment is performed by using a lentiviral vector constructed by the sequence of sgRNA 2.
1.2 construction of anti-MSLN-CAR vector:
the invention designs a mesothelin-targeting CAR vector (anti-MSLN-CAR) sequence, which comprises a CSF2R signal peptide, an extracellular segment (anti-MSLN scFv) for targeting and recognizing MSLN, a CD8 Hinge region, a CD8a transmembrane segment, a 4-1BB intracellular costimulatory signal domain and an intracellular signaling molecule CD3 zeta.
The Anti-MSLN-CAR gene full-length sequence is shown as SEQ ID NO:1 is shown in the specification;
ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTGATCCCCGACATCCAGATGGCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGCAGGTATCCTGCAGAGCATCTGGCTATAGTATCAATACTTACTATATGCAGTGGGTGCGGCAGGCCCCTGGAGCAGGCCTTGAGTGGATGGGCGTTATCAACCCCAGTGGTGTCACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACTTTGACCAACGACACGTCCACAAACACAGTCTACATGCAGTTGAACAGTCTGACATCTGCCGACACGGCCGTCTACTACTGTGCGAGATGGGCCTTATGGGGGGACTTCGGTATGGACGTCTGGGGCAAGGGAACCCTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACCATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGA(SEQ ID NO：1)。
the nucleotide sequence of the CSF2R signal peptide is shown as SEQ ID NO:2 is shown in the specification;
ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCTG(SEQ ID NO：2)。
the nucleotide sequence of the anti-MSLN single-chain antibody is shown as SEQ ID NO:3 is shown in the specification;
ATCCCCGACATCCAGATGGCCCAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAGTGCAGGTATCCTGCAGAGCATCTGGCTATAGTATCAATACTTACTATATGCAGTGGGTGCGGCAGGCCCCTGGAGCAGGCCTTGAGTGGATGGGCGTTATCAACCCCAGTGGTGTCACAAGTTACGCACAGAAGTTCCAGGGCAGAGTCACTTTGACCAACGACACGTCCACAAACACAGTCTACATGCAGTTGAACAGTCTGACATCTGCCGACACGGCCGTCTACTACTGTGCGAGATGGGCCTTATGGGGGGACTTCGGTATGGACGTCTGGGGCAAGGGAACCCTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTATTGGAGACAGAGTCACCATCACCTGCCGGGCCAGTGAGGGTATTTATCACTGGTTGGCCTGGTATCAGCAGAAGCCAGGGAAAGCCCCTAAACTCCTGATCTATAAGGCCTCTAGTTTAGCCAGTGGGGCCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAATATAGTAATTATCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAACGT(SEQ ID NO：3)。
SEQ ID NO:3 (SEQ ID NO: 11) the amino acid sequence of the anti-MSLN single-chain antibody encoded by SEQ ID NO:
IPDIQMAQVQLVQSGAEVKRPGASVQVSCRASGYSINTYYMQWVRQAPGAGLEWMGVINPSGVTSYAQKFQGRVTLTNDTSTNTVYMQLNSLTSADTAVYYCARWALWGDFGMDVWGKGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSTLSASIGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGAPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQYSNYPLTFGGGTKLEIKR(SEQ ID NO：11)；
the nucleotide sequence of the CD8 Hinge region is shown as SEQ ID NO:4 is shown in the specification;
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO：4)。
SEQ ID NO:4 (SEQ ID NO: 12) the amino acid sequence of the CD8 Hinge region encoded by SEQ ID NO:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO：12)；
the nucleotide sequence of the CD8 transmembrane region is shown as SEQ ID NO:5 is shown in the specification;
ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO：5)。
SEQ ID NO:5 (SEQ ID NO: 13) the amino acid sequence of the CD8 transmembrane region encoded by SEQ ID NO:
IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO：13)；
the nucleotide sequence of the 4-1BB co-stimulation region is shown as SEQ ID NO:6 is shown in the specification;
AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO：6)。
SEQ ID NO:6 (SEQ ID NO: 14) the amino acid sequence of the encoded 4-1BB costimulatory region is as follows:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO：14)；
the nucleotide sequence of the CD3 zeta intracellular region is shown as SEQ ID NO:7 is shown in the specification;
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO：7)。
SEQ ID NO:7 (SEQ ID NO: 15) the amino acid sequence of the intracellular domain of CD3 ζ encoded by SEQ ID NO:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGSVQPPRTPTTPFTCRPCPL(SEQ ID NO：15)；
the inventor uses pSBbi-MSLN CAR-GP plasmid as a template to amplify anti-MSLN-CAR gene fragment, inserts the anti-MSLN-CAR fragment between EcoRI and AsiSI enzyme cutting sites of lentiviral vector pCDH-CMV-MCS-EF1a-RFP, and constructs pCDH-CMV-anti-MSLN-CAR-EF1a-RFP vector.
2. Packaging of lentivirus and concentration of virus liquid
2.1 preparation of lentivirus packaging LentiCRISPR-NKG2A-KO and concentration of virus solution: taking 293T cells in logarithmic growth phase at 5X 10 6 Inoculating into 10cm culture dish, adding 10mL DMEM medium, and culturing at 37 deg.C with 5% CO 2 Was cultured overnight in an incubator. When the density of 293T cells reaches 80%, 10mL of fresh DMEM medium is replaced, and the cells are continuously placed at 37 ℃ and 5% CO 2 The incubator of (2) for cultivation. Preparing a lentivirus packaging system: adding 6 mu g of psPAX2 plasmid, 3 mu g of pMD2.G plasmid and 6 mu g of LentiCRISPR-NKG2A-KO plasmid into a serum-free DMEM culture medium with the volume of 250 mu L, and uniformly mixing to prepare a DNA mixed solution; mixing with 15 μ L
Adding into serum-free DMEM medium with the volume of 235 mu L, and uniformly mixing. Will be provided with
The mixed solution is added into the DNA mixed solution at one time, and is kept stand and mixed evenly, and is incubated for 15min at room temperature. The mixture was added to 293T cell culture dishes. And after 24h, liquid change is carried out. The culture dish was returned to 37 ℃ with 5% CO 2 In an incubator. After 48h, the cell supernatant was harvested, centrifuged at 400 Xg for 5min to remove cell debris, and the supernatant was filtered through a 0.45 μm filter tip into a new 50ml centrifuge tube. Add 5 XPEG 8000 solution, reverse the centrifuge tube upside down and mix well, put in 4 ℃ refrigerator overnight. Centrifuging at 4 deg.C and 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, subpackaging to EP tube to obtain virus concentrate, and storing in-80 deg.C refrigerator.
2.2 preparation of lentivirus packaging pCDH-CMV-anti-MSLN-CAR-EF1a-RFP and concentrate of virus: taking 293T cells in logarithmic growth phase at 5X 10 6 Inoculating into 10cm culture dish, adding 10mL DMEM medium, and culturing at 37 deg.C with 5% CO 2 Was cultured overnight in an incubator. When the 293T cell density reached 80%, 10mL of fresh DMEM medium was replaced, the mixture was further incubated at 37 ℃ and 5% CO 2 The incubator of (2) for cultivation. Preparing a lentivirus packaging system: adding 6 mu g of psPAX2 plasmid, 3 mu g of pMD2.G plasmid and 6 mu g of pCDH-CMV-anti-MSLN-CAR-EF1a-RFP plasmid into a serum-free DMEM culture medium with the volume of 250 mu L, and uniformly mixing to prepare a DNA mixed solution; mixing with 15 μ L
Adding into serum-free DMEM medium with the volume of 235 mu L, and uniformly mixing. Will->
The mixed solution is added into the DNA mixed solution at one time, and is kept stand and mixed evenly, and is incubated for 15min at room temperature. The mixture was added to 293T cell culture dishes. And after 24h, liquid change is carried out. The culture dish was returned to 37 ℃ with 5% CO 2 In an incubator. After 48h, the cell supernatant was harvested, centrifuged at 400 Xg for 5min to remove cell debris and the supernatant was filtered through a 0.45 μm filter tip into a new 50ml centrifuge tube. The virus solution was concentrated by adding 5 XPEG 8000 solution, mixing by turning the centrifuge tube upside down, and placing in a refrigerator at 4 ℃ overnight. Centrifuging at 4 deg.C and 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, subpackaging to EP tube to obtain virus concentrate, and storing in-80 deg.C refrigerator.
3. Lentiviral titer detection
293T cells in the logarithmic growth phase were collected, and the concentration of 293T cells was adjusted to 1X 10 5 and/mL. A24-well plate was prepared and 1mL of cell suspension (1X 10) was added to each well 5 Per well) a gradient of 3 cells was set. Placing at 37 ℃ and 5% CO 2 The incubator of (4) was cultured overnight. Firstly, diluting the concentrated virus solution obtained in the step 2: take 1.5mL of EP tube, aspirate 60. Mu.L of virus concentrate into EP tube, use540 μ L DMEM medium was diluted and mixed well. Changing culture medium, sucking 5 and 50 μ L virus liquid, adding to corresponding wells, marking, and returning the culture plate to 37 deg.C and 5% CO 2 The incubator of (1). And (5) collecting cells after 72h, carrying out flow detection on the 293T cell RFP expression rate, and converting the virus titer according to a formula.
Titer(TU/ml)＝100,000(target cells)×(％of GFP-positive cells/100)/volume of supernatant(in mL)。
4. Lentiviral infection of human NK cells
4.1 packaging of pCDH-CMV-anti-MSLN-CAR-EF1a-RFP lentivirus infection of human NK-92 cells: NK-92 cells in the logarithmic phase of growth were taken, and 2mL of α -MEM medium was added to resuspend the cells, thereby adjusting the cell density of NK-92 cells to 5X 10 5 one/mL. Cut-in 5X 10 in 24-well plate 5 NK-92 cells, 1. Mu.L protamine (final concentration 8. Mu.g/mL) and 1mL viral concentrate packaging pCDH-CMV-anti-MSLN-CAR-EF1a-RFP lentivirus obtained in step 2.2. Placing at 37 deg.C and 5% CO 2 Culturing in the incubator for 24h, observing the cell state, and changing the culture solution to obtain the infected cells. The infected cells were transferred into EP tubes, centrifuged at 100 Xg for 5min, resuspended in a small amount of fresh alpha-MEM medium, transferred to a cell culture flask, and cultured with 10mL of fresh alpha-MEM medium and IL-2 (final concentration of 200 IU/mL). After the infected cells were expanded, the infected cells were transferred into a flow tube, 3mL of 1 XPBS was added to resuspend the cells, centrifuged at 100 Xg for 5min, the supernatant was discarded, the cell pellet was flicked off, and this was repeated once. And (4) detecting the expression rate of NKG2A by a flow meter. And continuously expanding the culture to adjust the state of the NK-92 cells after infection. And (3) sorting the RFP positive NK-92 cells by using a sorting type flow cytometer to obtain anti-MSLN-CAR-NK-92 cells.
4.2 LentiCRISPR-NKG2A-KO packaging lentivirus infection anti-MSLN-CAR-NK-92 cells: taking anti-MSLN-CAR-NK-92 cells in the logarithmic growth phase, adding 2mL of alpha-MEM culture medium to resuspend the cells, and adjusting the cell density of the NK-92 cells to 5 × 10 5 one/mL. Inserting 5X 10 in 24-hole plate 5 Individual anti-MSLN-CAR-NK-92 cells, 1. Mu.L protamine (final concentration 8. Mu.g/mL) and 1mL of the package obtained in step 2.1Virus concentrate containing LentiCRISPR-NKG2A-KO lentivirus. Placing at 37 ℃ and 5% CO 2 Culturing in the incubator for 24h, observing the cell state, and changing the culture solution to obtain the infected cells. The infected cells were transferred into EP tubes, centrifuged at 100 Xg for 5min, a small amount of fresh α -MEM medium was added to resuspend the cells, the infected cells were transferred into cell culture flasks, and culture was continued by adding 10mL of fresh α -MEM medium and IL-2 (final concentration of 200 IU/mL). After the infected cells were expanded, the infected cells were transferred into a flow tube, 3ml of 1 × PBS was added to resuspend the cells, centrifuged for 5min at 100 × g, the supernatant was discarded, the cell pellet was flicked off, and this was repeated once. And (4) detecting the expression rate of the NKG2A by using a flow meter. And continuously expanding the culture to adjust the state of the anti-MSLN-CAR-NK-92 cells after infection. After the flow sorting, the purity of the anti-MSLN-CAR-NK-92 cells after infection is close to 99 percent, and NKG2A is obtained low anti-MALN CAR-NK-92 cell, CAR-NK cell for short.
Example 2: expression level and biological function identification of CAR-NK cell NKG2A
Expression levels of CAR-NK-92 cell NKG2A and expression of other inhibitory receptors
Flow cytometry is used for detecting and comparing the expression level of anti-MSLN CAR-NK-92 cell NKG2A before and after NKG2A is knocked down, the height of NK-92 cell expressing NKG2A can be seen, the positive rate is close to 100%, after CRISPR/Cas9 technology gene knockout, 80.4% of CAR-NK cell becomes CAR-NK cell of low expression NKG2A, and the specific reference is shown in figure 3.
Simultaneously observing whether the expression of other exhaustion-related molecules is influenced while the NKG2A is silenced, and respectively carrying out the treatment on NK-92 cells, CAR-NK-92 cells and NKG2A low and (3) incubating the anti-MALN CAR-NK-92 cells and ovarian cancer cells A1847 for 72 hours at a ratio of 1, collecting the NK cells, and carrying out flow detection on the expression of inhibitory receptors such as PD-1, tim-3 and TIGIT on the cell surface of the CAR-NK-92 cells. As a result, NKG2A was found to be comparable to CAR-NK-92 cell and NK-92 cell low The expression of PD-1 and Tim-3 on the cell surface of anti-MALN CAR-NK-92 is obviously reduced, and the expression of TIGIT is not obviously different, and particularly, the expression is shown in figure 4. Therefore, the results further show that the silencing of the expression of NKG2A on the surface of CAR-NK cells also weakens the expression of other inhibitory receptors such as PD-1, tim-3 and the like, and promotesThe balance of CAR-NK cell activating and inhibitory receptors is skewed towards activation analysis, thereby improving activation and function, and enhancing CAR-NK cell resistance to tumor microenvironment-induced depletion.
2. Expression of mesothelin and NKG2A ligand HLA-E in tumor cells
The expression of Mesothelin (MSLN) on the cell surface of human ovarian cancer cell lines a1847, HO8910 and a2780 was examined by flow cytometry and the results are shown in figure 5. The results showed that a1847 cells expressed high levels of mesothelin (97.2% positive rate), hey cells expressed medium levels (32.8% positive rate), and HO8910 and a2780 cells expressed low levels of mesothelin with 1.83% and 0.56% positive rates, respectively.
The research shows that the NKG2A ligand HLA-E is highly expressed on the surfaces of ovarian cancer tissues and various ovarian cancer cells. The inventors examined the expression of HLA-E on the cell surface of human ovarian cancer cell lines A1847, HO8910 and A2780 by flow cytometry, and the results are shown in FIG. 6. The results showed that HO8910 cells expressed high levels of HLA-E (positive rate of 95.5%), A1847 cells expressed medium levels (positive rate of 36.0%), and A2780 cells expressed low levels of HLA-E (positive rate of 17.4%). Therefore, the inventors selected HO8910 and A1847 cells as HLA-E positive target cells and A2780 cells as HLA-E negative target cells.
In vitro killing of NK-92 cells after NKG2A knockdown
NK-92, anti-MSLN CAR-NK-92, NKG2A low anti-NSLN CAR-NK-92 is effector cells, ovarian cancer cell lines A1847, HO8910 and A2780 are target cells, the effective target ratio is set to be 10. The results show that when the effective target ratio is 10, NKG2A low The killing efficiency of anti-NSLN CAR-NK-92 cells to A1847 cells which moderately express NKG2A ligand HLA-E is 63.77 +/-4.39 percent, which is obviously higher than that of anti-MSLN CAR-NK-92 group (49.77 +/-2.78 percent) and NK-92 group (37.60 +/-1.50 percent); NKG2A low The killing efficiency of anti-NSLN CAR-NK-92 on HO8910 cells highly expressing HLA-E is 52.10 +/-4.52%, which is obviously higher than that of anti-MSLN CAR-NK-92 group (32.70 +/-2).0%) and NK-92 group (30.20 ± 0.95%); and the killing efficiency of the three groups of effector cells to the A2780 cells with low expression of HLA-E is not obviously different. Therefore, the results show that the knocking-down of NKG2A can obviously improve the killing capacity of CAR-NK cells on HLA-E positive tumor cells.
In addition, the inventor also detects the condition that whether the NKG2A is knocked down or not and NK cells kill and are associated with degranulation. Each effector cell was incubated with ovarian cancer cells for 5h at an effective target ratio of 10, and cell culture supernatants were collected and assayed for levels of secretion of granzyme B (granzyme B) and perforin (perforin) by ELISA, see fig. 8. As a result, NKG2A was incubated with HLA-E-moderately expressing A1847 cells and HLA-E-highly expressing HO8910 cells low anti-NSLN CAR-NK-92 cells secreted granzyme B and perforin at levels significantly higher than both CAR-NK-92 and NK-92 groups. However, there was no significant difference in the level of granzyme B and perforin secretion by the groups of effector cells after co-incubation with HLA-E-overexpressing A2780 cells. Further verifies that the knocking-down of NKG2A can obviously improve the degranulation level and the killing function of CAR-NK cells on HLA-E positive tumor cells.
IFN-gamma secretion level of NK-92 cells after NKG2A knockdown
Detecting the IFN-gamma secretion capacity change of NK-92 cells after NKG2A knockdown by ELISA technology, and detecting the NK cells (NK-92, anti-MSLN CAR-NK-92 and NKG 2A) low anti-NSLN CAR-NK-92) were incubated with ovarian cancer cells for 5h, respectively, at a target-to-effect ratio of 10. As a result, NKG2A was incubated with HO8910 cells highly expressing HLA-E low The level of IFN-gamma expression of anti-NSLN CAR-NK-92 cells is obviously higher than that of anti-MSLN CAR-NK92 group and NK-92 group; NKG2A after Co-incubation with HLA-E-moderately expressing A1847 cells low anti-NSLN CAR-NK-92 cells expressed IFN-gamma at levels significantly higher than in NK-92 group. However, the level of IFN-gamma secretion did not change significantly in any of the groups after co-incubation with HLA-E-overexpressing A2780 cells. Therefore, the experiment shows that the knocking-down of NKG2A can obviously improve the IFN-gamma secretion capacity of CAR-NK cells when the CAR-NK cells are contacted with HLA-E positive tumor cells.
Examples3：NKG2A low CAR-NK cell in vivo antitumor capability and tumor-bearing mouse survival period
Carrying out subcutaneous tumor loading on ovarian cancer A1847 cells with positive mesothelin and HLA-E, establishing an ovarian cancer xenograft model, and observing the treatment effect of the CAR-NK cells with low NKG2A on ovarian cancer. Selecting 4-week-old female nude mice, and carrying tumor under axilla at a dose of 2 × 10 per mouse 6 A1847 cells, after one week, the tumor volume reached 100mm 3 Treatment was started on the left and right, see fig. 10 in particular. Mice were first randomly divided into control group, NK-92 cell treatment group, anti-MSLN CAR-NK-92 cell treatment group, and NKG2A low anti-MSLN CAR-NK-92 cell treatment group. The mice in the treatment group are injected with effector cells by tail vein at 1X 10 7 Control group was injected with an equal volume of 1 XPBS solution every other week and every 3 days with IL-2 (5X 10) 4 IU/only). The tumor volume of the mice was measured every three days, and tumor growth curves and mouse survival curves were plotted, see fig. 11 and 12.
The results showed that all treatment groups significantly inhibited tumor growth compared to the tumor-bearing control group, whereas NKG2A was administered low The anti-MSLN CAR-NK-92 cell therapy group had the best therapeutic effect and the tumor volume was significantly smaller than CAR-NK-92 and NK-92 groups (FIG. 11). Furthermore, NKG2A low The anti-MSLN CAR-NK-92 cell treatment group significantly prolonged the survival of mice and increased the survival of mice compared to CAR-NK-92 and NK-92 groups (FIG. 12). Therefore, the experiments show that the CAR-NK cells with the knocked-down NKG2A have an in-vivo tumor killing effect, can resist the inhibition effect of a tumor microenvironment, resist functional depletion and play a strong anti-tumor role.
In the description of the specification, reference to the description of "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" or the like means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A CAR-immune cell, wherein the NKG2A gene of the CAR-immune cell is silenced;
wherein the immune cells of the CAR-immune cells comprise at least one of NK cells, T cells, NKT cells, and γ δ T cells.
2. The CAR-immune cell of claim 1, wherein the immune cell of the CAR-immune cell is an NK cell;
optionally, the NKG2A gene is silenced by knocking out the NKG2A gene of the CAR-immune cell.
3. The CAR-immune cell of claim 2, wherein the knockout is achieved by a CRISPR/Cas9 system;
optionally, the sgRNA of the CRISPR/Cas9 system has the sequence as set forth in SEQ ID NO: at least one of the nucleotide sequences shown in 8-10;
optionally, the knockout is performed by:
introducing an expression vector carrying the sgRNA and a nucleic acid encoding the Cas9 molecule into CAR-immune cells to be modified for culture treatment;
optionally, the time of the culture treatment is 24-72 h;
optionally, the expression vector is a eukaryotic cell expression vector.
4. The CAR-immune cell of any one of claims 1 to 3, wherein the CAR of the CAR-immune cell comprises:
an extracellular region, wherein the extracellular region comprises a single-chain antibody and a hinge region, the C end of the single-chain antibody is connected with the N end of the hinge region, and the single-chain antibody specifically recognizes a tumor antigen;
a transmembrane region, wherein the N end of the transmembrane region is connected with the C end of the hinge region of the extracellular region;
an intracellular domain, wherein the N-terminus of the intracellular domain is linked to the C-terminus of the transmembrane domain;
optionally, the antigen comprises at least one selected from the group consisting of MSLN, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, epCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, BCMA;
preferably, the antigen is MSLN.
5. The CAR-immune cell of claim 4, wherein the single chain antibody has an amino acid sequence as set forth in SEQ ID NO. 11;
optionally, the hinge region is selected from the hinge region of a CD8 molecule;
optionally, the hinge region has an amino acid sequence as shown in SEQ ID NO 12.
6. The CAR-immune cell of claim 4, wherein the transmembrane region is selected from a CD8 molecular transmembrane segment;
optionally, the transmembrane region has an amino acid sequence shown as SEQ ID NO. 13.
7. The CAR-immune cell of claim 4, wherein the intracellular region comprises a costimulatory domain and an intracellular signaling domain;
optionally, the co-stimulatory domain is selected from the intracellular segment of the 41BB molecule;
optionally, the co-stimulatory domain has an amino acid sequence as shown in SEQ ID No. 14;
optionally, the intracellular signaling domain is selected from the intracellular segment of the CD3 ζ molecule;
optionally, the intracellular signaling domain has an amino acid sequence as set forth in SEQ ID NO 15.
8. A pharmaceutical composition, comprising:
the CAR-immune cell of any one of claims 1 to 7; and
optionally a pharmaceutically acceptable carrier or adjuvant.
9. Use of a CAR-immune cell according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 in the manufacture of a medicament for eliminating or reducing immune escape mechanisms of a tumor.
10. Use of a CAR-immune cell according to any of claims 1 to 7 or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the prevention and/or treatment of a tumor or cancer;
preferably, the tumor is a solid tumor or a hematological tumor.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211504757.5A CN115948341A (en) | 2022-11-28 | 2022-11-28 | CAR-immunocyte for knocking down NKG2A gene and application thereof |
| PCT/CN2023/095928 WO2024113714A1 (en) | 2022-11-28 | 2023-05-24 | Car-immune cell for knocking down nkg2a gene and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211504757.5A CN115948341A (en) | 2022-11-28 | 2022-11-28 | CAR-immunocyte for knocking down NKG2A gene and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115948341A true CN115948341A (en) | 2023-04-11 |
Family
ID=87290431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211504757.5A Pending CN115948341A (en) | 2022-11-28 | 2022-11-28 | CAR-immunocyte for knocking down NKG2A gene and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN115948341A (en) |
| WO (1) | WO2024113714A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820645A (en) * | 2022-11-28 | 2023-03-21 | 上海恩凯细胞技术有限公司 | Method for preparing NK (natural killer) cells capable of silencing NKG2A genes and application of NK cells |
| WO2024113714A1 (en) * | 2022-11-28 | 2024-06-06 | 上海恩凯细胞技术有限公司 | Car-immune cell for knocking down nkg2a gene and use thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110079529A (en) * | 2019-04-28 | 2019-08-02 | 成都美杰赛尔生物科技有限公司 | SgRNA, expression vector, kit and application thereof for targeting knockout people's NKG2A/KLRC1 gene |
| US20230310600A1 (en) * | 2020-08-07 | 2023-10-05 | Crage Medical Co., Limited | Engineered cells and method for engineering cells |
| JP2023552998A (en) * | 2020-12-03 | 2023-12-20 | ンカルタ・インコーポレイテッド | Methods of engineering immune cells for enhanced efficacy and persistence, and the use of engineered cells in immunotherapy |
| WO2022242700A1 (en) * | 2021-05-20 | 2022-11-24 | Wuxi Biologics (Shanghai) Co., Ltd. | Genetically modified nk cells and uses thereof |
| WO2022242701A1 (en) * | 2021-05-20 | 2022-11-24 | Wuxi Biologics (Shanghai) Co., Ltd. | Genetically modified gamma-delta t cells and uses thereof |
| CN115948341A (en) * | 2022-11-28 | 2023-04-11 | 上海恩凯细胞技术有限公司 | CAR-immunocyte for knocking down NKG2A gene and application thereof |
-
2022
- 2022-11-28 CN CN202211504757.5A patent/CN115948341A/en active Pending
-
2023
- 2023-05-24 WO PCT/CN2023/095928 patent/WO2024113714A1/en unknown
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115820645A (en) * | 2022-11-28 | 2023-03-21 | 上海恩凯细胞技术有限公司 | Method for preparing NK (natural killer) cells capable of silencing NKG2A genes and application of NK cells |
| CN115820645B (en) * | 2022-11-28 | 2023-09-22 | 上海恩凯细胞技术有限公司 | Method for preparing NK cells silencing NKG2A gene and application thereof |
| WO2024113714A1 (en) * | 2022-11-28 | 2024-06-06 | 上海恩凯细胞技术有限公司 | Car-immune cell for knocking down nkg2a gene and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024113714A1 (en) | 2024-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11427810B2 (en) | Modified oncolytic virus | |
| CN115948341A (en) | CAR-immunocyte for knocking down NKG2A gene and application thereof | |
| WO2019243847A1 (en) | Treatment using oncolytic virus | |
| US20180221463A1 (en) | Modified NK Cells and Uses Thereof | |
| CN115838439B (en) | Preparation method and application of chimeric transition receptor gene modified NK cells | |
| WO2024113777A1 (en) | Transgenic immune cell and use thereof | |
| WO2024113649A1 (en) | Method for improving viability and anti-tumor activity of nk cells and use thereof | |
| WO2024119768A1 (en) | Method for preparing nk cells capable of reversing inhibitory signal from tumor microenvironment and application | |
| CN116064620A (en) | Preparation and application of CAR-NK cells for enhancing infiltration capacity to tumor parts | |
| WO2024113715A1 (en) | Method for preparing nk cells for silencing nkg2a gene and use thereof | |
| CN115103912B (en) | Plasmid combination and application thereof in preparation of modified immune cells | |
| CN104906575A (en) | Application of LSECtin as melanoma immunotherapy target | |
| CN115066248A (en) | Chimeric antigen receptor dendritic cells (CAR-DCs) and methods of making and using same | |
| CN115232217B (en) | SynNotch structure and application thereof | |
| CN118048312A (en) | CD19 and/or CD20 targeted CAR-NK cells and preparation and application thereof | |
| CN116376920A (en) | Preparation method and application of CEACAM5 targeted CAR-T cells | |
| CN117304342A (en) | Chimeric antigen receptor and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40086762 Country of ref document: HK |