PD-1 gene knockout MUC1-targeting CAR-T cell as well as preparation method and application of PD-1 gene knockout MUC1-targeting CAR-T cell
A technology of PD-1 and gene knockout, which is applied in the field of CAR-T cells and its preparation, can solve problems such as side effects and destruction of immune tolerance, and achieve the effects of high specificity, improved curative effect, and wide indications
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Examples
Embodiment 1
[0071] Example 1 Preparation of Lentivirus Loaded with CAR
[0072] 1. Construction of lentiviral expression vector pLVX-EF1α-CAR 5E5
[0073] The CAR structure consists of an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, and an intracellular signaling domain. The nucleotide sequence of CAR in this example is shown in SEQ ID NO: 10, provided by Suzhou Artificially synthesized by Synbio Biotechnology Co., Ltd. and cloned into the lentiviral vector pLVX-EF1α-IRES-Puro (purchased from Shanghai Linyuan Biotechnology Co., Ltd.) between the EcoRI and MluI restriction sites to construct a recombinant lentivirus The expression vector is named pLVX-EF1α-CAR 5E5, and the plasmid map is as follows: figure 1 shown.
[0074] Plasmid extraction was performed with an endotoxin-free plasmid maxi kit (Endo-free Plasmid Maxi Kit, Omega). At the same time, a large number of lentiviral packaged helper vector plasmids (pSPAX2 and pMD2.G) were extracted. The...
Embodiment 2
[0080] Example 2 Preparation of CAR-T cells
[0081] (1) Take 50 mL of peripheral blood from tumor patients or healthy volunteers, anticoagulate with heparin, and centrifuge the obtained serum, inactivate at 56°C for later use.
[0082] (2) Precipitated cells were diluted with normal saline and added to a centrifuge tube filled with Ficoll solution (purchased from GE), separated by density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMC), washed with normal saline 2 times, count to spare.
[0083] (3) Resuspend PBMC with lymphocyte medium KBM 581 serum-free cell medium (Corning), and adjust the cell density to 1-2×10 6 / mL, inoculate into T75 cell culture flask, add anti-human CD3 monoclonal antibody (OKT-3) to activate PBMC, supplement 500IU / mL recombinant human interleukin-2 (rhIL-2) at the same time, 5~10% plasma, in 37°C, 5% CO 2 Cultured in an incubator to obtain activated T cells.
[0084] (4) Take T cells activated overnight, add purified ...
Embodiment 3
[0088] Example 3 CAR-T cell PD-1 gene knockout
[0089] (1) Take the CAR-T cells infected with the lentivirus in Example 2 for 2-3 days, wash 3 times with OPTI-MEM (Thermo) and resuspend with OPTI-MEM, the cell density is 0.5-1x10 8 cells / mL.
[0090] (2) During the centrifugation process, the purified Cas9 protein and PD-1-gRNA (GenScript Biotechnology Co., Ltd.) (SEQ ID NO: 11 and SEQ ID NO: 52) were mixed and incubated at room temperature for 15 minutes to obtain Cas9 RNP (Cas9 ribonucleoprotein).
[0091] Experimental group: 100 μL of cell suspension and Cas9 RNP were mixed, added to a 2 mm electric shock cup, and electroporated and transfected by BTX ECM830 (Harvard).
[0092] Control group: 100 μL of cell suspension and Cas9 protein were mixed, added to a 2 mm electric shock cup, and electroporated and transfected by BTX ECM830 (Harvard).
[0093] (3) Quickly transfer the cells to a 6-well plate with 2 mL of 37°C preheated KBM581 medium (containing 500 U / mL rhIL-2) at...
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Description
Claims
Application Information
- IPC
- C07K19/00; C12N15/62; C12N5/10; C12N15/867; A61K39/00; A61P35/00
- CPC
- C07K16/3092; C07K14/7051; C12N5/0636; C07K14/70521; C12N15/86; A61K39/00117; A61P35/00; C07K2319/02
- Inventors
- 周超; 安鸿