WO2020000526A1 - Lymphocyte t shp-1-inactivé et procédé de construction associé - Google Patents

Lymphocyte t shp-1-inactivé et procédé de construction associé Download PDF

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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
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cells
car
shp
gene
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胡边
张琳琳
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Aomiao Biotechnology (guangzhou) Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—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
    • 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
    • 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.

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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.
PCT/CN2018/095635 2018-06-26 2018-07-13 Lymphocyte t shp-1-inactivé et procédé de construction associé Ceased WO2020000526A1 (fr)

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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

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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

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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
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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

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