WO2020000526A1 - Lymphocyte t shp-1-inactivé et procédé de construction associé - Google Patents
Lymphocyte t shp-1-inactivé et procédé de construction associé Download PDFInfo
- Publication number
- WO2020000526A1 WO2020000526A1 PCT/CN2018/095635 CN2018095635W WO2020000526A1 WO 2020000526 A1 WO2020000526 A1 WO 2020000526A1 CN 2018095635 W CN2018095635 W CN 2018095635W WO 2020000526 A1 WO2020000526 A1 WO 2020000526A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- car
- shp
- gene
- cell
- Prior art date
Links
- 238000010276 construction Methods 0.000 title abstract description 6
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 90
- 101710128901 Tyrosine-protein phosphatase non-receptor type 6 Proteins 0.000 claims abstract description 50
- 102100021657 Tyrosine-protein phosphatase non-receptor type 6 Human genes 0.000 claims abstract description 21
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims description 42
- 108091033409 CRISPR Proteins 0.000 claims description 28
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 14
- 101000610551 Homo sapiens Prominin-1 Proteins 0.000 claims description 13
- 102100040120 Prominin-1 Human genes 0.000 claims description 13
- 230000014509 gene expression Effects 0.000 claims description 11
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 9
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 9
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 9
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 9
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 claims description 9
- 102100025096 Mesothelin Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 210000004881 tumor cell Anatomy 0.000 claims description 8
- 101150029707 ERBB2 gene Proteins 0.000 claims description 7
- 230000030279 gene silencing Effects 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 6
- 108020004414 DNA Proteins 0.000 claims description 5
- 108700005075 Regulator Genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- -1 BMSA Proteins 0.000 claims description 4
- 230000002147 killing effect Effects 0.000 abstract description 9
- 238000009169 immunotherapy Methods 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 description 15
- 230000000694 effects Effects 0.000 description 12
- 238000004520 electroporation Methods 0.000 description 8
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 7
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 6
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 6
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000010354 CRISPR gene editing Methods 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 101150058049 car gene Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000003209 gene knockout Methods 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
- 238000012546 transfer Methods 0.000 description 4
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 3
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 3
- 229940045513 CTLA4 antagonist Drugs 0.000 description 3
- 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
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 230000022534 cell killing Effects 0.000 description 3
- 238000002038 chemiluminescence detection Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000004068 intracellular signaling Effects 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 description 2
- 238000012815 AlphaLISA Methods 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
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 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
- 208000008839 Kidney Neoplasms 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
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 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
- 108700010039 chimeric receptor Proteins 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000005746 immune checkpoint blockade Effects 0.000 description 2
- 229940126546 immune checkpoint molecule Drugs 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 108010005465 AC133 Antigen Proteins 0.000 description 1
- 102000005908 AC133 Antigen Human genes 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
- 239000012275 CTLA-4 inhibitor Substances 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
- 230000007018 DNA scission Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 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
- 101000818543 Homo sapiens Tyrosine-protein kinase ZAP-70 Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 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
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 239000012270 PD-1 inhibitor Substances 0.000 description 1
- 239000012668 PD-1-inhibitor Substances 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 101150005990 SHP1 gene Proteins 0.000 description 1
- 230000006044 T cell activation Effects 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
- 230000002411 adverse Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009175 antibody therapy 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
- 230000005540 biological transmission Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect 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
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000033607 mismatch repair Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940121655 pd-1 inhibitor Drugs 0.000 description 1
- 229960002621 pembrolizumab Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000017105 transposition 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
- 230000003313 weakening effect Effects 0.000 description 1
Images
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
Definitions
- the invention relates to the technical fields of tumor immunology, molecular biology, cell biology and gene editing, and in particular relates to a method for preparing SHP-1 knockout T cells by using a CRISPR / Cas9 system.
- Immune checkpoint blocking therapy has shown amazing results in the treatment of tumors. Clinical trials have found that immune checkpoint blocking therapy can reactivate the effector functions of depleted T cells and increase the anti-tumor activity of T cells. Immune checkpoint blocking therapy mainly uses antibodies to block the interaction between immune checkpoint molecules and their corresponding antibodies to improve the anti-tumor ability of T cells. The most common immune checkpoint molecules are PD-1 and CTLA-4. Immune checkpoint blocking therapy has achieved significant results in the treatment of a variety of tumors, including melanoma, non-small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, and mismatch repair Defective tumors [1].
- CTLA-4-targeting antibody ipilimumab has been approved for use in the treatment of patients with advanced melanoma, 20% of whom have received antibody therapy and have a survival period of more than three years.
- PD-1 antibodies nivolumab and pembrolizumab and PD-L1 antibody atezolizumab have also been approved by the FDA for the treatment of advanced melanoma, non-small cell lung cancer and kidney cancer.
- Some patients show partial or complete remission after receiving immune checkpoint blockade. However, not all patients respond to the treatment of CTLA-4 and PD-1 inhibitors.
- T cells The presence of phosphatase in T cells has important regulatory effects on the function of T cells.
- T cells recognize the antigen through the TCR-MHC complex, a large number of intracellular signaling molecules will be phosphorylated and activated by kinases such as Lck, ZAP70, and participate in signal transmission.
- kinases such as Lck, ZAP70
- a large number of inhibitory phosphatases in T cells will also be activated, causing dephosphorylation of signal molecules and inactivation, affecting the transmission of T cell activation signals, and ultimately weakening T cells. Killing function of cells [2].
- PTP phosphatase is an intracellular protein, its function cannot be blocked by antibody blocking agents such as PD-1 and CTLA-4; small molecule inhibitors of phosphatase often lack the ability to inhibit enzymes or cells. Specificity, leading to great clinical side effects. These reasons also increase the difficulty for people to make choices and operations.
- T cells are terminally differentiated primary cells, and it is difficult to perform genetic manipulations such as transfection or infection, coupled with their limited proliferation capacity, which limits the genetic manipulation of T cells in vitro, especially gene knockout.
- gene editing technology has provided an opportunity for T cell immunotherapy.
- Gene editing with CRISPR / Cas9 technology is simple, efficient, and specific, which makes gene editing of T cells in vitro gradually possible from possible.
- the purpose of the present invention is to overcome the shortcomings of the prior art and provide an SHP-1 knockout T cell and a method for constructing the same.
- Another object of the present invention is to provide a SHP-1 knockout CAR-T cell and a method for constructing the same.
- the T cells of tumor cells are efficiently killed, and the SHP-1 gene of T cells is endogenously silenced.
- the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out.
- the third exon of the SHP-1 gene of the T cells was knocked out.
- the T cells are CAR-T cells.
- CAR-T cells include CD133 CART, CD19 CART, CD20 CART, BMSA CART, MSLN CART, EGFRVIII CART, Her2 CART, GD2 CART, CEA CART.
- the method for constructing the T cell includes introducing into the T cell a plasmid DNA expressing Cas9 and sgRNA, a Cas9 protein or a sgRNA-Cas9 protein complex, and knocking out at least a part of the sequence of SHP-1 or at least a part of its expression control sequence to make SHP-1 Gene endogenous silencing.
- the sgRNA sequence targets the third exon of the SHP-1 gene.
- CAR-T cells include CD19 CART, CD20 CART, BMSACART, MSLN CART, EGFRVIIICART, Her2CART, GD2CART, CEACART.
- a method for treating a tumor includes the following steps:
- the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out.
- the CAR modification is selected from the group consisting of CD133, CD19, CD20, BMSA, MSLN, EGFRVIII, Her2, GD2, and CEA modifications.
- the invention breaks through the limitations of the prior art, creatively blocks SHP-1 in T cells endogenously, and effectively improves the killing effect of T cells on tumors.
- CAR-T cells such as 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 SHP in CAR T cells -1 knockout can effectively improve the ability of CAR-T cells to kill tumor cells and provide a new target for tumor immunotherapy.
- Cas9 and sgRNA-expressing plasmids were transferred to T cells to achieve efficient knockout of the SHP-1 gene. Compared with the previously reported use of Cas9 protein or Cas9 mRNA and electrotransduction of sgRNA transcribed in vitro , Saving time and cost of preparation.
- FIG. 1 is a structure of a third-generation CAR used in the present invention, including a CSF2RA chimeric receptor signal peptide, an extracellular antigen-binding region (scFv), a c-Myc tag peptide, a CD8 hinge region, and an intracellular signaling region;
- a CSF2RA chimeric receptor signal peptide an extracellular antigen-binding region (scFv), a c-Myc tag peptide, a CD8 hinge region, and an intracellular signaling region;
- FIG. 2 shows the knockout of the SHP-1 gene in T cells after T cells were transfected by the plasmid expressing Cas9 and sgRNA in Example 1 of the present invention
- 3 is the result of overexpression of CD133 CAR and knockout of the SHP-1 gene in T cells by plasmid electrotransformation in the present invention, and the expression of the CAR gene and the knockout of the SHP-1 gene in the T cells were detected, respectively;
- Figure 4 is a comparison of the efficiency of different sgRNAs
- FIG. 5 is a detection of the ability to kill tumor cells and detection of cytokine secretion by SHP-1 knockout CD133 CAR-T cells prepared in Example 3 of the present invention
- Figure 6 shows the results of the safety experiment of SHP-1 gene knockout in vitro mediated by CRISPR / Cas9.
- the T cells of tumor cells are efficiently killed, and the SHP-1 gene of T cells is endogenously silenced.
- Gene silencing refers to a decrease in the expression level of a gene or a lack of expression. Endogenous silencing means that the expression of the gene itself is reduced or is not expressed.
- the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out. So that SHP-1 cannot be expressed normally, or active SHP-1 cannot be obtained after expression.
- CAR-T cells have specific lethality to specific cells.
- the T cells are CAR-T cells.
- CAR-T cells include but are 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 method for constructing the T cell includes introducing into the T cell a plasmid DNA expressing Cas9 and sgRNA, a Cas9 protein or a sgRNA-Cas9 protein complex, and knocking out at least a part of the sequence of SHP-1 or at least a part of its expression control sequence to make SHP-1 Gene endogenous silencing.
- the sgRNA sequence targets the third exon of the SHP-1 gene.
- CAR-T cells include CD19 CART, CD20 CART, BMSACART, MSLN CART, EGFRVIIICART, Her2CART, GD2CART, CEACART.
- Figure 1a shows the position of the sgRNA-targeted sequence on the SHP1 gene.
- T7EN1 digestion showed (Figure 1b) that cleavage of DNA occurred at the sgRNA targeting site.
- Sanger sequencing results showed ( Figure 1c) that the mutation rate of SHP-1 gene was 87.5% (sevenths of eight).
- the CAR-targeted antigen used in this example is CD133, and the structure of the CAR is shown in Figure 2. In this order, it includes the CSF2RA chimeric receptor signal peptide (SEQ ID NO: 1), the extracellular antigen-binding region (scFv, SEQ ID:
- PBMCs were activated with human anti-CD3 / CD28 and Dynabeads, and cultured in AIM-V medium containing 10% FBS and 300U / ml IL-2. After 5 days of cell activation, Dynabeads were removed and cultured, and 0.5 ⁇ g / ml puromycin drug sieve to enrich CAR positive T cells;
- the inventors also selected sgRNAs targeted at other sites for comparison ( Figure 4a and Figure 4b).
- T7EN1 to detect and compare the cleavage effect of different sgRNAs
- the sg1 used above has the best effect
- the other sgRNA sg2- 4
- the editing efficiency is lower than sg1 (editing efficiency is proportional to the brightness of the smaller bands generated, Figure 4b).
- the experimental data show that the sequence selected and optimized in the present invention has better targeting and cutting efficiency, and has unexpected effects.
- the method used for cell killing detection is the Luciferase-Luciferin chemiluminescence detection method. Luciferase is overexpressed in the target cells for killing. After T cells are killed, the number of target cells is reduced and the chemiluminescence detection value is reduced. By comparing the chemiluminescence detection values of target cells before and after killing, the killing ability of T cells is estimated.
- the target cells used in the present invention are U251 cells (U251-CD133-luc) that overexpress CD133 antigen and Luciferase, and the non-target cells are U251 cells (U251-CD133) that overexpress luciferase.
- the specific operations are as follows:
- T cells un-transduced T cells
- CD133 CAR CAR T cells
- SHP-1 KO CD133 CAR gene-edited CAR T cells
- the cultured cells were collected, washed once with PBS, and resuspended in 100 ⁇ l of PBS. The cells were transferred to an opaque 96-well plate, 100 ⁇ l of D-luciferin at a concentration of 150 ⁇ g / ml was added, and the luminal at 560 nm was detected by a microplate reader;
- T cell electroporation uses plasmid electrotransformation, which may cause random insertion of the genome. Simultaneous insertion of Cas9 and sgRNA will cause multiple cuts of the genome and improve off-target effects. Because the Cas9 protein is fused with a GFP fluorescent protein, the integration of Cas9 can be roughly judged by detecting whether there is a GFP signal. The insertion of Cas9 was detected by flow cytometry one day and 24 days after electric rotation ( Figure 6a). The results showed that no Cas9 gene was integrated into the genome.
- CAR T cells can be constructed in a similar way without creative effort.
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
La présente invention concerne un lymphocyte t SHP-1-inactivé et un procédé de construction associé L'effet destructeur du lymphocyte T sur des tumeurs peut être efficacement amélioré par le blocage endogène de SHP -1 dans le lymphocyte T, fournissant ainsi une nouvelle cible pour l'immunothérapie antitumorale.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810665548.6A CN108866004A (zh) | 2018-06-26 | 2018-06-26 | Shp-1敲除的t细胞及其构建方法 |
CN201810665548.6 | 2018-06-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020000526A1 true WO2020000526A1 (fr) | 2020-01-02 |
Family
ID=64294683
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2018/095635 WO2020000526A1 (fr) | 2018-06-26 | 2018-07-13 | Lymphocyte t shp-1-inactivé et procédé de construction associé |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN108866004A (fr) |
WO (1) | WO2020000526A1 (fr) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116334077B (zh) * | 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) | 一种气道上皮细胞shp-1基因特异性敲除小鼠模型的构建方法及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016154596A1 (fr) * | 2015-03-25 | 2016-09-29 | Editas Medicine, Inc. | Procédés, compositions et constituants liés à crispr/cas |
CN106163547A (zh) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | 使用嵌合抗原受体治疗癌症 |
CN107109421A (zh) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | Car表达载体及car表达t细胞 |
WO2018096361A1 (fr) * | 2016-11-28 | 2018-05-31 | Autolus Limited | Protéine modifiant la transduction de signal |
CN108138183A (zh) * | 2014-04-18 | 2018-06-08 | 爱迪塔斯医药公司 | 用于癌症免疫疗法的crispr-cas相关方法、组合物和组分 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107164407A (zh) * | 2017-07-04 | 2017-09-15 | 王小平 | 无物种限制的真核生物同时进行基因敲除和基因过表达 |
-
2018
- 2018-06-26 CN CN201810665548.6A patent/CN108866004A/zh active Pending
- 2018-07-13 WO PCT/CN2018/095635 patent/WO2020000526A1/fr active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106163547A (zh) * | 2014-03-15 | 2016-11-23 | 诺华股份有限公司 | 使用嵌合抗原受体治疗癌症 |
CN108138183A (zh) * | 2014-04-18 | 2018-06-08 | 爱迪塔斯医药公司 | 用于癌症免疫疗法的crispr-cas相关方法、组合物和组分 |
CN107109421A (zh) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | Car表达载体及car表达t细胞 |
WO2016154596A1 (fr) * | 2015-03-25 | 2016-09-29 | Editas Medicine, Inc. | Procédés, compositions et constituants liés à crispr/cas |
WO2018096361A1 (fr) * | 2016-11-28 | 2018-05-31 | Autolus Limited | Protéine modifiant la transduction de signal |
Non-Patent Citations (1)
Title |
---|
WATSON, H. A. ET AL.: "SHP-1: the next checkpoint target for cancer immunotherapy", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 44, no. 2, 15 April 2016 (2016-04-15), pages 356 - 362, XP055469547, DOI: 10.1042/BST20150251 * |
Also Published As
Publication number | Publication date |
---|---|
CN108866004A (zh) | 2018-11-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Mathewson et al. | Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis | |
Ding et al. | PARP inhibition elicits STING-dependent antitumor immunity in Brca1-deficient ovarian cancer | |
US10034925B2 (en) | Modified natural killer cells and natural killer cell lines having increased cytotoxicity | |
Li et al. | Anti-cancer efficacy of SREBP inhibitor, alone or in combination with docetaxel, in prostate cancer harboring p53 mutations | |
Mandula et al. | Ablation of the endoplasmic reticulum stress kinase PERK induces paraptosis and type I interferon to promote anti-tumor T cell responses | |
Park et al. | Generation of lung cancer cell lines harboring EGFR T790M mutation by CRISPR/Cas9-mediated genome editing | |
CN111148518A (zh) | 使用cdk4/6抑制剂调控调节性t细胞和免疫应答的方法 | |
US20220235380A1 (en) | Immune cells having co-expressed shrnas and logic gate systems | |
CN107699547A (zh) | Pd‑1基因沉默的靶向cd133的car t细胞及其应用 | |
Gjuka et al. | Enzyme-mediated depletion of methylthioadenosine restores T cell function in MTAP-deficient tumors and reverses immunotherapy resistance | |
WO2020000526A1 (fr) | Lymphocyte t shp-1-inactivé et procédé de construction associé | |
Yang et al. | Targeting RAD51 enhances chemosensitivity of adult T‑cell leukemia‑lymphoma cells by reducing DNA double‑strand break repair | |
Miyauchi et al. | Reprogramming of tumor-associated macrophages via NEDD4-mediated CSF1R degradation by targeting USP18 | |
Li et al. | Aurora A kinase inhibition induces accumulation of SCLC tumor cells in mitosis with restored interferon signaling to increase response to PD-L1 | |
Morman et al. | BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice | |
Ho et al. | The CD58: CD2 axis is co-regulated with PD-L1 via CMTM6 and governs anti-tumor immunity | |
CN114929853A (zh) | 用于治疗胶质母细胞瘤和其他癌症的天然杀伤细胞免疫疗法 | |
CN115348870B (zh) | 通用型嵌合抗原受体t细胞及其应用 | |
Kinsella et al. | Attenuation of homeostatic signaling from apoptotic thymocytes triggers a global regenerative response in the thymus | |
CN114949218B (zh) | 一种pd-l1调控剂及其应用 | |
Jacob et al. | Gene therapy in the treatment and prevention of oral cancer: An overview | |
US20220304990A1 (en) | Mi-2beta Inhibitor as an Immunotherapy Agent | |
EP3635098B1 (fr) | Lymphocytes t modifiés pour surexprimer lephf19 | |
Bag et al. | The immunomodulatory properties of the HDAC6 inhibitor ACY241 supports robust anti-tumor response in NSCLC when coupled with the chemotherapy drug Oxaliplatin | |
Mengwasser | Genetic screening approaches to cancer driver characterization and synthetic lethal target discovery |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18924341 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 19.05.2021) |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 18924341 Country of ref document: EP Kind code of ref document: A1 |