CN108866004A - The T cell and its construction method that SHP-1 is knocked out - Google Patents
The T cell and its construction method that SHP-1 is knocked out Download PDFInfo
- Publication number
- CN108866004A CN108866004A CN201810665548.6A CN201810665548A CN108866004A CN 108866004 A CN108866004 A CN 108866004A CN 201810665548 A CN201810665548 A CN 201810665548A CN 108866004 A CN108866004 A CN 108866004A
- Authority
- CN
- China
- Prior art keywords
- cell
- car
- shp
- gene
- sgrna
- 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
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 82
- 101710128901 Tyrosine-protein phosphatase non-receptor type 6 Proteins 0.000 title claims abstract description 48
- 102100021657 Tyrosine-protein phosphatase non-receptor type 6 Human genes 0.000 title claims abstract description 27
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims description 41
- 108091033409 CRISPR Proteins 0.000 claims description 28
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 24
- 239000013612 plasmid Substances 0.000 claims description 16
- 108020004414 DNA Proteins 0.000 claims description 11
- 230000002147 killing effect Effects 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 230000008685 targeting Effects 0.000 claims description 8
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 7
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 7
- 101150029707 ERBB2 gene Proteins 0.000 claims description 7
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 7
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 7
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 claims description 7
- 102100025096 Mesothelin Human genes 0.000 claims description 7
- 210000004881 tumor cell Anatomy 0.000 claims description 7
- 108010085220 Multiprotein Complexes Proteins 0.000 claims description 3
- 102000007474 Multiprotein Complexes Human genes 0.000 claims description 3
- 230000033228 biological regulation Effects 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 11
- 230000000903 blocking effect Effects 0.000 abstract description 8
- 238000009169 immunotherapy Methods 0.000 abstract description 2
- 230000001665 lethal effect Effects 0.000 abstract description 2
- 230000005611 electricity Effects 0.000 description 21
- 230000000694 effects Effects 0.000 description 15
- 230000006872 improvement Effects 0.000 description 12
- 238000011282 treatment Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 8
- 230000022534 cell killing Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000010362 genome editing Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000010354 CRISPR gene editing Methods 0.000 description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 5
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 4
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 4
- 229940045513 CTLA4 antagonist Drugs 0.000 description 4
- 101150058049 car gene Proteins 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 238000007480 sanger sequencing Methods 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 3
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 238000012815 AlphaLISA Methods 0.000 description 2
- 108091007741 Chimeric antigen receptor T cells Proteins 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
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000031146 intracellular signal transduction Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- 108010005465 AC133 Antigen Proteins 0.000 description 1
- 102000005908 AC133 Antigen Human genes 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000818543 Homo sapiens Tyrosine-protein kinase ZAP-70 Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 244000134336 Malus baccata Species 0.000 description 1
- 235000005079 Malus baccata Nutrition 0.000 description 1
- 239000012270 PD-1 inhibitor Substances 0.000 description 1
- 239000012668 PD-1-inhibitor Substances 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 102000002727 Protein Tyrosine Phosphatase Human genes 0.000 description 1
- 101150005990 SHP1 gene Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 102000008579 Transposases Human genes 0.000 description 1
- 108010020764 Transposases Proteins 0.000 description 1
- 102100021125 Tyrosine-protein kinase ZAP-70 Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960003852 atezolizumab Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 229940126546 immune checkpoint molecule Drugs 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000033607 mismatch repair Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 229940121655 pd-1 inhibitor Drugs 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 108020000494 protein-tyrosine phosphatase Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- 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
- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- 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
- C12N2510/00—Genetically modified 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses the SHP-1 T cell knocked out and its construction methods.Inventors have found that can effectively improve T cell by blocking the SHP-1 endogenous in T cell to the lethal effect of tumour, new target spot provided for tumour immunotherapy.
Description
Technical field
The present invention relates to tumor immunology, molecular biology, cell biology and gene editing technical fields, more particularly to
A method of T cell is knocked out using CRISPR/Cas9 system preparation SHP-1.
Background technique
Immunologic test point blocking treatment shows surprising curative effect in the treatment of tumour.Clinical test discovery, is immunized inspection
The effector function for exhausting T cell can be reactivated by making an inventory of blocking treatment, improve the anti-tumor activity of T cell.Immunologic test point
Blocking treatment mainly improves T cell using antibody come interaction between blocking immunity checkpoint molecule and its corresponding antibodies
Anti-tumor capacity.The most common immunologic test point molecule is PD-1 and CTLA-4.Immunologic test point blocking treatment is more
Remarkable effect is obtained in the treatment of kind tumour, including melanoma, lung cancer in non-cellule type, Hodgkin lymphoma, neck
Cancer, oophoroma, kidney, the tumour [1] of bladder cancer and mis-match repair deficient.Target the antibody ipilimumab of CTLA-4
In the granted treatment for advanced melanoma patient, wherein 20% received the patient of Antybody therapy, life cycle 3 years with
On.Recently, the antibody atezolizumab of the antibody nivolumab and pembrolizumab and PD-L1 of PD-1 are also obtained
FDA ratifies, in the treatment for advanced melanoma, lung cancer in non-cellule type and kidney.Some patientss are receiving immune inspection
After making an inventory of blocking treatment, show part and alleviate either complete incidence graph, however, not all patient to CTLA-4 and
The treatment of PD-1 inhibitor has reaction.
The presence of phosphatase has important adjustment effect to the Function of T cell in T cell.T cell passes through TCR-
After MHC compound identifies antigen, a large amount of signaling molecules intracellular can be activated by tyrosine phosphorylations such as Lck, ZAP70, participate in letter
Number transmitting.However the excessive activation in order to maintain stable state, prevent T cell, largely inhibit acid phosphatase that can also be swashed in T cell
It is living, it causes the dephosphorylation of signaling molecule and inactivates, affect the transmitting of t cell activation signal, finally weaken killing for T cell
Hurt function [2].There are more than 100 kinds of PTP phosphatases in cell, at least 45 kinds is contained human T cells, and more and more
Experimental evidence show these phosphatases often and have the function of independence and can not be substituted.Therefore, although theoretically suitably inhibiting T
The activity of phosphatase is possible to that the ability that T cell removes tumour cell can be improved in cell, but likewise, improper inhibition T is thin
The activity of phosphatase in born of the same parents, the activity of especially crucial phosphatase, may result in unforeseeable negative consequence, to siberian crabapple
The influence of system is unknown, or even causes the generation of autoimmune disease.Selecting any target spot as us therein is also
One problem [2] recognized altogether.In addition, since PTP phosphatase is intracellular protein, it can not be by resisting as PD-1, CTLA-4
Body blocking agent realizes the blocking to its function;The micromolecular inhibitor of phosphatase also tends to lack to the special of enzyme or cell
Property, cause clinically toxic side effect it is very big.These reasons also both increase the difficulty that people select and operate.
T cell is the primary cell of terminal differentiation, is difficult the genetic manipulations such as to be transfected or infected, along with it is proliferated energy
Power is limited, and which limits the knockouts for carrying out genetic manipulation, especially gene to T cell in vitro.In recent years, gene editing skill
The revolutionary development of art provides opportunity for T cell immunization therapy.Gene editing, which is carried out, using CRISPR/Cas9 technology operates letter
Single, high-efficient, high specificity, this to carry out gene editing gradually by that may come true to T cell in vitro.
Bibliography:
1.Topalian SL,Drake CG,Pardoll DM.(2015)Immune checkpoint blockade:a
common denominator approach to cancer therapy.Cancer Cell.27(4):450-461
2.Mustelin T,Vang T,Bottini N.(2005)Protein tyrosine phosphatases and
the immune response.Nat Rev Immunol.5(1):43-57.。
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of SHP-1 T cell knocked out and its building sides
Method.
It is another object of the present invention to provide CAR-T cells and its construction method that a kind of SHP-1 is knocked out.
The technical solution adopted by the present invention is that:
The T cell of Efficient killing effect tumour cell, the SHP-1 gene of T cell is by silent endogenous.
As the further improvement of above-mentioned T cell, the SHP-1 gene or its controlling gene of T cell are at least partly knocked out.
As the further improvement of above-mentioned T cell, the 3rd exon of the SHP-1 gene of T cell is knocked.
As the further improvement of above-mentioned T cell, T cell is CAR-T cell.
As the further improvement of above-mentioned T cell, CAR-T cell includes CD133 CAR T, CD19 CAR T, CD20
CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
The construction method of above-mentioned T cell, Plasmid DNA from expression Cas9 and sgRNA to T cell, Cas9 albumen including importing
Or sgRNA-Cas9 protein complexes, knock out SHP-1 at least partly sequence or its at least partly expression regulation sequence makes SHP-1
Gene silent endogenous.
The further improvement of construction method as above-mentioned T cell, sgRNA sequence target aobvious outside the 3rd of SHP-1 gene
Son.
The further improvement of construction method as above-mentioned T cell, CAR-T cell include CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
The beneficial effects of the invention are as follows:
The present invention breaks through the limitation of the prior art, creatively blocks the SHP-1 endogenous in T cell, effectively improves
Lethal effect of the T cell to tumour.Particularly, by CAR-T cell, such as CD133 CAR-T, CD19 CAR T, CD20 CAR
In T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T cell
SHP-1 is knocked out, and can effectively improve the ability of CAR-T cell killing tumour cell, new target is provided for tumour immunotherapy
Point.
By turning by Plasmid DNA electricity, the plasmid electricity for expressing Cas9 and sgRNA is gone in T cell, is realized efficient
The knockout of SHP-1 gene carries out electricity by Cas9 albumen or Cas9 mRNA and the sgRNA of in-vitro transcription with what is reported before
Phase inversion ratio has saved time and the cost of preparation.
Detailed description of the invention
Fig. 1 is the structure for the three generations CAR that the present invention uses, including CSF2RA Chimerical receptor signal peptide, after birth exoantigen knot
Close area (scFv), c-Myc label peptide fragment, CD8 hinge area, intracellular signal transduction area;
Fig. 2 is SHP-1 in T cell after turning T cell by the plasmid electricity of expression Cas9 and sgRNA in the embodiment of the present invention 1
The knockout situation of gene;
Fig. 3 is that by plasmid electricity turn, realization is overexpressed CD133 CAR and knocks out SHP-1 gene in T cell in the present invention
As a result, having detected the knockout situation of the expression of CAR gene and SHP-1 gene in T cell respectively;
Fig. 4 is that the efficiency of different sgRNA compares;
The CD133 CAR-T cell killing tumour cell ability that Fig. 5 knocks out for SHP-1 obtained in the embodiment of the present invention 3
Detection and cytokine secretion profile detection;
Fig. 6 is the safety experiment result for the cell in vitro SHP-1 gene knockout that CRISPR/Cas9 is mediated.
Specific embodiment
The T cell of Efficient killing effect tumour cell, the SHP-1 gene of T cell is by silent endogenous.
Gene silencing refers to the expression quantity decline of gene or does not express substantially.Silent endogenous refers to the expression quantity of gene itself
Decline is not expressed substantially.
As the further improvement of above-mentioned T cell, the SHP-1 gene or its controlling gene of T cell are at least partly knocked out.
So that SHP-1 can not also be unable to get active SHP-1 after normal expression, or expression.
As the further improvement of above-mentioned T cell, the 3rd exon of the SHP-1 gene of T cell is knocked.Test number
It according to showing to be knocked out for this exon, has higher efficiency, targeting is more preferable, and correspondingly safety is also more preferably.
CAR-T cell has specific killing power to specific cell.As the further improvement of above-mentioned T cell, T cell is
CAR-T cell.
As the further improvement of above-mentioned T cell, CAR-T cell includes but is not limited to CD133 CAR T, CD19 CAR
T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA
CAR T etc..
The construction method of above-mentioned T cell, Plasmid DNA from expression Cas9 and sgRNA to T cell, Cas9 albumen including importing
Or sgRNA-Cas9 protein complexes, knock out SHP-1 at least partly sequence or its at least partly expression regulation sequence makes SHP-1
Gene silent endogenous.
The further improvement of construction method as above-mentioned T cell, sgRNA sequence target aobvious outside the 3rd of SHP-1 gene
Son.
The further improvement of construction method as above-mentioned T cell, CAR-T cell include CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
Below with reference to embodiment and experiment, further exemplary illustration technical solution of the present invention.Other CAR T cells
It knocks out and is referred to following method progress.
Embodiment 1
Turn the knockout that SHP-1 gene is realized in T cell by Cas9 albumen and sgRNA electricity
1) experiment PBMC needs first to activate via human anti-CD3/CD28Dynabeads, and culture is containing 10%
In the AIM-V culture medium of FBS and 300U/ml IL-2;
2) Dynabeads is removed two days later and carries out electricity turns:
3) sgRNA for taking 4 μ g to be transcribed in vitro and 16 μ g Cas9 albumen mix, and are incubated at room temperature 20 minutes;
4) 82 μ l electricity are added into above-mentioned system to turn buffer (Lonza, V4XP-3024), 18 μ l supplement 1,
It mixes;
5) it after taking 1million PBMC cell, PBS to clean twice, is resuspended with above-mentioned system, and be transferred in electric revolving cup,
Turned using Lonza-4D electroporation EO-115 program electricity;
6) electricity turns to terminate that cell is transferred in the culture medium preheated in advance immediately to cultivate.
7) electricity turns counting in latter 10 days, and carries out T7EN1 digestion identification and the determining knockout efficiency of Sanger sequencing.
As a result as shown in Figure 1.Fig. 1 a shows position of the sequence of sgRNA targeting on SHP1 gene.T7EN1 digestion
It shows (Fig. 1 b), the cutting of DNA has occurred in sgRNA target site.Sanger sequencing result shows (Fig. 1 c), the base of SHP-1
Because mutation rate is 87.5% (7/8ths).
Embodiment 2:
Turn to realize in T cell by plasmid electricity and is overexpressed CD133CAR and knockout SHP-1 gene.
1) antigen for the CAR targeting that the present embodiment uses is CD133, and the structure of CAR is as shown in Fig. 2, successively include
CSF2RA Chimerical receptor signal peptide (SEQ ID NO:1), after birth exoantigen combined area (scFv, SEQ ID NO:
2), c-Myc label peptide fragment (SEQ ID NO:3), CD8 hinge area, intracellular signal transduction area (SEQ ID NO:
4) the resistant gene puromycin connected with T2A small peptide.Entire CAR gene is loaded in PiggyBac plasmid,
Constitute PiggyBac-CD133 CAR.The expression of CAR gene initial gene under the driving of EF1 α promoter.CAR gene integration
What is relied on into genome is the transposition of transposons;
2) by PiggyBac-CD133 CAR plasmid, transposase plasmid, Cas9 plasmid (Addgene plasmid#
44758) and knock out SHP-1 gene the sgRNA plasmid (sequence of sgRNA such as SEQ ID NO:Shown in 5) each 5 μ g and Amaxa electricity
Turn the electricity in kit and turn reagent mixing, and is resuspended 2 × 10 with this mixed liquor7A PBMC cell is simultaneously transferred in electric revolving cup, benefit
Turned with Lonza-2B electroporation U-014 program electricity;
3) electricity turns to terminate that cell is transferred in the culture medium preheated in advance immediately to cultivate;
4) after one day, activate PBMC using human anti-CD3/CD28 Dynabeads, culture containing 10%FBS and
In the AIM-V culture medium of 300U/ml IL-2, after cell-stimulating 5 days, removes Dynabeads and continue to cultivate, and with 0.5 μ g/ml
Puromycin medicine sieve, be enriched with the CAR positive T cell;
5) it when fortnight, determines by T7EN1 digestion identification and Sanger sequencing and knocks out efficiency;It is examined by flow cytometer showed
Survey the expression of CAR.
As a result as shown in Figure 3.The sgRNA sequence knocked out for SHP-1 is consistent with Fig. 1 a.T7EN1 digestion shows (Fig. 3 a),
The cutting of DNA has occurred in sgRNA target site.Sanger sequencing result shows (Fig. 3 b) that the gene mutation rate of SHP-1 is
87.5% (7/8ths).The expression of CAR is in knockout group and does not knock out in group in 95% or more (Fig. 3 c).
Opposite with above-mentioned sgRNA used, inventor has selected the sgRNA of some other site targetings to compare simultaneously
(Fig. 4 a and Fig. 4 b).When being detected using T7EN1 digestion and the cutting effect of more different sgRNA, it can be found that above-mentioned used
Sg1 effect it is best, and other the 2nd exons of targeting or are equally the sgRNA (sg2-4) of the 3rd exon, editor
Efficiency is lower than sg1 (editorial efficiency is directly proportional to the brightness of smaller band generated, Fig. 4 b).Experimental data shows this hair
The bright middle sequence after selecting, optimizing has better targeting and shear efficiency, has unexpected effect.
Embodiment 3:
Experiment in vitro proves the fragmentation effect for the CD133 CAR-T cells against tumor that SHP-1 is knocked out.
By killing experiments in vitro, cytokine secretion experiments have shown that SHP-1 is knocked out to CD133 CAR-T cell anti-tumor
The influence of ability.
The method that cell killing detection uses is Luciferase-Luciferin chemiluminescence detecting method, and killing target is thin
Luciferase is overexpressed in born of the same parents, after T cell killing, target cell population is reduced, and the luminous detected value of induced chemical reduces.Pass through ratio
Compared with the chemiluminescence detection value before killing with target cell after killing, the killing ability of T cell is estimated.The target cell that the present invention uses
For the U251 cell (U251-CD133-luc) for being overexpressed CD133 antigen and Luciferase, non-target cell is to be overexpressed
The U251 cell (U251-CD133) of luciferase.Concrete operations are as follows:
1) 2 × 10 are taken5A target cell or non-target cell are laid in 48 orifice plates;
2) according to effect target than 1:Isosorbide-5-Nitrae:1,16:1 ratio is separately added into the T cell (T cell) that electricity does not turn over, CAR T
The CAR T cell (SHP-1KO CD133 CAR) that cell (CD133CAR) and gene editing are crossed, wherein it is thin that T is not added in control group
Born of the same parents' culture, 300 hole μ l/ of total volume, every kind of effect target are put in 37 DEG C, 5%CO than three multiple holes of setting2Culture in cell incubator
16-18 hours;Microscope, which is taken pictures, detects T cell killing ability;
3) cell of culture is collected, after PBS cleaning is primary, with 100 μ l PBS resuspension.And cell is transferred to opaque
In 96 orifice plates, the D-luciferin that 100 μ l concentration are 150 μ g/ml is added, microplate reader detects Luminal 560nm luminous value;
4) the killing ability of T cell is calculated:Using the hole for containing only PBS as blank control, reads as N0, contain only tumour
Reading is set as N in the hole of cell, and experiment group number-reading is set as N1, then cell killing efficiency is 1- (N1-N0)/(N-N0) × 100%.
Cytokine release experiment is detected using AlphaLISA reagents series box:
1) by 2 × 105A target cell U251-CD133-luc is laid in 48 orifice plates, is 16 according to effect target ratio:1 ratio,
The CAR-T cell that a certain amount of CAR-T cell or SHP-1 are knocked out is added into every hole, every kind of T cell is divided into three multiple holes, altogether
300 μ l AIM-V culture solutions are added to cultivate in 37 DEG C of incubators;
2) after 16 hours, 100 μ l supernatants are drawn, 500g is centrifuged 5 minutes, is collected supernatant and is used for subsequent detection.It uses
The AlphaLISA reagents series box detection IFN-γ and TNF-α cytokine release situation of PerkinElmer company.
As a result as shown in Figure 5.SHP-1 knock out after significantly enhance CAR T cell killing tumor cell ability (Fig. 5 a,
5b), the ability (Fig. 5 c) of CAR T cell secrete cytokines TNF-α and IFN-γ is enhanced.
Embodiment 4:
In vitro cell experiment proves the safety for the SHP-1 gene knockout that CRISPR/Cas9 is mediated.
T cell electricity turns the form turned using plasmid electricity, is likely to result in the radom insertion of genome, Cas9 and sgRNA's
Insertion will cause the multiple cutting of genome simultaneously, improve undershooting-effect.Because Cas9 protein fusion has GFP fluorescin, lead to
Cross the integration for detecting whether that GFP signal can substantially judge Cas9.Turn to turn with electricity one day after by flow cytometer detection electricity
The insertion situation (Fig. 6 a) of 24 days Cas9 afterwards.The result shows that and Cas9 gene integration has been not detected into genome.
Another safety, that is, mop effect of CRISPR/Cas9 system progress gene editing.According to targeting SHP-1's
SgRNA sequence finds the highest site of missing the target of marking on the website www.crispr.mit.edu.It is final to choose preceding ten marking
Highest site, which is used as, potentially misses the target site (Fig. 6 b).Further potentially missed the target site by PCR and T7EN1 to first four
It is detected.T7EN1 restriction analysis shows (Fig. 6 c), does not occur undershooting-effect in corresponding site.Experimental data table
Bright system of the invention has good safety.
Other CAR T cells can also use similar method, and building obtains without creative efforts.
SEQUENCE LISTING
<110>Profound biotechnology(Guangzhou)Co., Ltd
<120>The T cell and its construction method that SHP-1 is knocked out
<130>
<140> CN2018106655486
<141> 2018-06-26
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 66
<212> DNA
<213>The mankind
<400> 1
atgctgctgc tggtcacttc tctgctgctg tgcgaactgc cccaccccgc ctttctgctg 60
attccc 66
<210> 2
<211> 744
<212> DNA
<213>The mankind
<400> 2
caggtccagc tgcagcagtc tggagctgag ctggtcagac ccggcgcatc agtgaaactg 60
agctgcaagg cttccggcta tactttctcc gactttgaga tgcactgggt caagcagacc 120
ccagtgcatg gcctggaatg gatcggggac attgatcccg gcactgggga caccgcctat 180
aacctgaagt tcaaaggcaa ggctaccctg accacagata agagctcctc tacagcctac 240
atggagctga ggtctctgac tagtgaagat tcagcagtct actattgcac actgggggcc 300
ttcgtgtact ggggacaggg cacactggtc accgtgagcg ccgctaaaac tacccccaag 360
ctggaggaag gagagttcag cgaagcaaga gtggacgtgg tcgtgaccca gacacccctg 420
tctctgcctg tcagttttgg cgatcaggtg agcatctcct gtaggagttc acagtcactg 480
gccaacagct acgggaatac atatctgtct tggtacctgc acaagccagg acagagtccc 540
cagctgctga tctatgggat ttccaatcgc ttctctggag tgcctgaccg attttctggg 600
agtggatcag gcaccgattt cacactgaaa atcagcacca ttaagcccga ggacctgggc 660
atgtactatt gtctgcaggg gacccatcag ccttacactt ttggcggggg aaccaaactg 720
gagatcaagc gagcagacgc agcg 744
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
gaacagaagc tcataagcga agaa 24
<210> 4
<211> 849
<212> DNA
<213>Artificial plasmid
<400> 4
ttcgtccccg tgttcctgcc tgccaagcca acaactaccc ctgctccacg accacctact 60
ccagcaccta ccatcgcaag tcagcccctg tcactgcgac ctgaggcttg ccggccagca 120
gctggaggag cagtgcacac ccgaggcctg gacttcgcat gcgatatcta catttgggca 180
ccactggctg gaacctgtgg ggtcctgctg ctgagcctgg tcatcaccct gtattgtaac 240
cacagaaata ggagcaaacg ctcccgactg ctgcattccg actacatgaa catgacacct 300
cggagaccag gccccactag aaagcattac cagccatatg ccccacccag ggatttcgca 360
gcctatcgga gccggttcag cgtcgtgaaa agggggcgca agaaactgct gtacatcttc 420
aagcagcctt ttatgcgccc agtgcagaca actcaggagg aagacggatg ctcttgtcgg 480
ttcccagagg aggaggaagg aggctgcgag ctgagagtga agttcagccg gagcgccgat 540
gcaccagcat atcagcaggg acagaatcag ctgtacaacg agctgaatct gggcaggcgc 600
gaggaatatg acgtgctgga taagcgacga ggacgggacc ccgaaatggg aggaaaaccc 660
agaaggaaga accctcagga ggggctgtat aatgaactgc agaaagacaa gatggctgag 720
gcatacagcg aaattggaat gaaaggagag cgccgacggg ggaagggaca cgatgggctg 780
taccagggac tgtcaaccgc cactaaagat acctacgacg cactgcacat gcaggctctg 840
cccccaaga 849
<210> 5
<211> 358
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (259)..(277)
<223> n is a, c, g, t or u
<400> 5
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg tgagaccgnn nnnnnnnnnn nnnnnnngtt ttagagctag aaatagcaag 300
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttt 358
Claims (8)
1. the T cell of Efficient killing effect tumour cell, it is characterised in that:The SHP-1 gene of T cell is by silent endogenous.
2. T cell according to claim 1, it is characterised in that:The SHP-1 gene or its controlling gene of T cell are by least
Part knocks out.
3. T cell according to claim 1, it is characterised in that:3rd exon of the SHP-1 gene of T cell is struck
It removes.
4. described in any item T cells according to claim 1~3, it is characterised in that:T cell is CAR-T cell.
5. T cell according to claim 4, it is characterised in that:CAR-T cell includes CD133 CAR T, CD19 CAR
T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA
CAR T。
6. the construction method of T cell described in claim 1, including imported to T cell expression Cas9 and sgRNA Plasmid DNA,
Cas9 albumen or sgRNA-Cas9 protein complexes knock out at least partly sequence or its at least partly expression regulation sequence of SHP-1
Column make SHP-1 gene silent endogenous.
7. according to the method described in claim 6, it is characterized in that:3rd exon of sgRNA sequence targeting SHP-1 gene.
8. method according to claim 6 or 7, it is characterised in that:CAR-T cell includes CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810665548.6A CN108866004A (en) | 2018-06-26 | 2018-06-26 | The T cell and its construction method that SHP-1 is knocked out |
PCT/CN2018/095635 WO2020000526A1 (en) | 2018-06-26 | 2018-07-13 | Shp-1-knockout t-cell and construction method therefor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810665548.6A CN108866004A (en) | 2018-06-26 | 2018-06-26 | The T cell and its construction method that SHP-1 is knocked out |
Publications (1)
Publication Number | Publication Date |
---|---|
CN108866004A true CN108866004A (en) | 2018-11-23 |
Family
ID=64294683
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810665548.6A Pending CN108866004A (en) | 2018-06-26 | 2018-06-26 | The T cell and its construction method that SHP-1 is knocked out |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN108866004A (en) |
WO (1) | WO2020000526A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116334077A (en) * | 2022-07-29 | 2023-06-27 | 江苏省人民医院(南京医科大学第一附属医院) | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107109421A (en) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | CAR expression vectors and CAR expression T cells |
CN107164407A (en) * | 2017-07-04 | 2017-09-15 | 王小平 | Gene knockout and gene overexpression are carried out simultaneously without the eucaryote that species are limited |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170335281A1 (en) * | 2014-03-15 | 2017-11-23 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
EP3132030B1 (en) * | 2014-04-18 | 2020-08-26 | Editas Medicine, Inc. | Crispr-cas-related methods, compositions and components for cancer immunotherapy |
EP3274454B1 (en) * | 2015-03-25 | 2021-08-25 | Editas Medicine, Inc. | Crispr/cas-related methods, compositions and components |
GB201620070D0 (en) * | 2016-11-28 | 2017-01-11 | Autolus Ltd | Signal transduction modifying protein |
-
2018
- 2018-06-26 CN CN201810665548.6A patent/CN108866004A/en active Pending
- 2018-07-13 WO PCT/CN2018/095635 patent/WO2020000526A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107109421A (en) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | CAR expression vectors and CAR expression T cells |
CN107164407A (en) * | 2017-07-04 | 2017-09-15 | 王小平 | Gene knockout and gene overexpression are carried out simultaneously without the eucaryote that species are limited |
Non-Patent Citations (1)
Title |
---|
INGUNN M.STROMNES等: "Abrogation of SHP-1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo", 《J IMMUNOL.》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116334077A (en) * | 2022-07-29 | 2023-06-27 | 江苏省人民医院(南京医科大学第一附属医院) | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
CN116334077B (en) * | 2022-07-29 | 2024-02-02 | 江苏省人民医院(南京医科大学第一附属医院) | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
Also Published As
Publication number | Publication date |
---|---|
WO2020000526A1 (en) | 2020-01-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Solomon et al. | Cohesin gene mutations in tumorigenesis: from discovery to clinical significance | |
US20200317777A1 (en) | Enhanced chimeric antigen receptors and uses thereof | |
CN106399306B (en) | Target sgRNA, genophore and its application that people lncRNA-UCA1 inhibits bladder cancer | |
CN109715803A (en) | Allele editor and its application | |
AU2018342245A1 (en) | Methods of isolating T cells having antigenic specificity for a p53 cancer-specific mutation | |
US20230105008A1 (en) | Methods and compositions for identifying castration resistant neuroendocrine prostate cancer | |
CN109517820A (en) | A kind of gRNA and HPK1 gene editing method targeting HPK1 | |
Zhang et al. | Downstream class switching leads to IgE antibody production by B lymphocytes lacking IgM switch regions | |
Solé et al. | AT follicular helper cell origin for T regulatory type 1 cells | |
CN115697356A (en) | Methods of treating cancer by inhibiting CARM1 | |
CN108866004A (en) | The T cell and its construction method that SHP-1 is knocked out | |
CN110172089A (en) | A kind of more antigen combinations of KRAS mutation, targeting KRAS mutation tumour CTL and its application | |
US20100267145A1 (en) | Immunocompetent cell having anti-cd38 antibody on its cell surface | |
WO2019036043A2 (en) | A method to generate a cocktail of personalized cancer vaccines from tumor-derived genetic alterations for the treatment of cancer | |
WO2011097618A1 (en) | Loss of de novo dna methyltransferases promotes expansion of normal hematopoietic stem cells | |
Mueller et al. | Targeting the mevalonate or Wnt pathways to overcome CAR T-cell resistance in TP53-mutant AML cells | |
Kwok et al. | Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens | |
Dikiy et al. | Terminal differentiation and persistence of effector regulatory T cells essential for the prevention of intestinal inflammation | |
CN107082811B (en) | Composite protein of chimeric antigen receptor and fusion induced apoptosis enzyme | |
Wang et al. | High recallability of memory B cells requires ZFP318-dependent transcriptional regulation of mitochondrial function | |
CN117866906B (en) | Use of FOXR A inhibitor in preparation of medicament for treating tumor | |
Zahedimaram | The identification of the mechanisms of lrba deficiency dependent defects in regulatory t-cell function | |
EP3635098B1 (en) | T-cells modified to overexpress phf19 | |
US20240091259A1 (en) | Generation of anti-tumor t cells | |
RU2804664C2 (en) | Hpv-specific binding molecules |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181123 |
|
RJ01 | Rejection of invention patent application after publication |