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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

Pending Publication Date: 2021-06-11
GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to overcome the influence of immunosuppression, PD-1 / PD-L1 antibodies have achieved good results in immunotherapy of solid tumors. However, long-term use of immune checkpoint inhibitors may destroy immune tolerance and cause serious side effects

Method used

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  • 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
  • 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
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a PD-1 gene knockout MUC1-targeting CAR-T cell as well as a preparation method and application thereof. An MUC1-targeting chimeric antigen receptor is prepared firstly, wherein the MUC1-targeting chimeric antigen receptor comprises an antigen binding structural domain, a transmembrane structural domain, a co-stimulation structural domain and an intracellular signal transduction structural domain. Tn glycosylation MUC1 is used as a target spot, the target spot specificity is high, off-target is not likely to occur, and the safety of the CAR-T cells is improved. On the basis, the PD-1 gene knockout MUC1-targeting CAR-T cell is obtained, and after the PD-1 gene is knocked out and the CAR-T cell is transfused back into a body, the CAR-T cell failure and inactivation caused by PD-L1 expressed by tumors cannot occur, so that the efficient specific cell killing effect on tumor cells is achieved, and the curative effect of the CAR-T cell is improved.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy, and in particular relates to a PD-1 gene knockout CAR-T cell targeting MUC1 and a preparation method and application thereof. Background technique [0002] Tumor treatment is one of the biggest problems plaguing human beings, and conventional treatment methods have their own limitations. With the advancement of science and technology, tumor immunotherapy is gradually becoming the development direction of tumor treatment, known as the fourth major tumor treatment technology. Tumor immunotherapy mainly includes adoptive cellular immunotherapy (ACT) represented by CAR-T (Chimeric Antigen Receptor T-Cell) and immune checkpoint therapy represented by PD-1 / PDL-1 antibody, which have made breakthroughs in recent years sexual progress. [0003] Chimeric Antigen Receptor (CAR) is an artificial receptor that mimics the function of TCR, which is composed of antigen recognition domain, hinge...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00
CPCC07K16/3092C07K14/7051C12N5/0636C07K14/70521C12N15/86A61K39/00117A61P35/00C07K2319/02C07K2319/03C07K2319/33C12N2510/00A61K2039/5158C12N2740/15043C12N2800/107
Inventor 周超安鸿周玲尹海滨
Owner GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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