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Chimeric antigen receptor, NKG2D CAR-NK cell expressing chimeric antigen receptor, preparation method and application thereof

A chimeric antigen receptor, antigen technology, applied in genetically modified cells, DNA/RNA fragments, cells modified by introducing foreign genetic material, etc. problem, to achieve the effect of low cost, less toxic and side effects, and improved targeting

Active Publication Date: 2019-07-19
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ligands of NKG2D molecules are not expressed or expressed very little in normal cells, but when cells are infected or cancerous, the expression of these ligands will increase significantly
[0005] At present, CAR-NK cells for the treatment of NKG2D-expressing tumors and related diseases do not yet exist. Only CAR-T cells are used for the treatment of NKG2D-expressing tumors and related diseases. NKG2D CAR-NK cells are bound to promote progress in the field of tumor therapy

Method used

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  • Chimeric antigen receptor, NKG2D CAR-NK cell expressing chimeric antigen receptor, preparation method and application thereof
  • Chimeric antigen receptor, NKG2D CAR-NK cell expressing chimeric antigen receptor, preparation method and application thereof
  • Chimeric antigen receptor, NKG2D CAR-NK cell expressing chimeric antigen receptor, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0078] The preparation of embodiment 1 lentiviral vector

[0079] Gene synthesis NKG2D-CD8 TM - 4-1BB-CD3ζ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 1, and the gene sequence is shown in SEQ ID NO: 2), through enzyme digestion, NKG2D-CD8 TM - The 4-1BB-CD3ζ fusion gene sequence was transformed and connected to the PRRSLIN vector, and the upstream of the gene was the EP-1α promoter. Transform the Stbl3 E. coli strain with the vector, screen with ampicillin, obtain positive clones, extract the plasmids, identify the clones by enzyme digestion, and obtain the PRRLSIN-NKG2D lentiviral transfection vector (such as figure 1 shown).

Embodiment 2

[0080] The preparation of embodiment 2 lentiviruses

[0081] (1) 24 hours before transfection, use about 8×10 per dish 6 293T cells were inoculated into 15cm culture dishes. Make sure that the cells are at about 80% confluence during transfection, and the transfected cells are evenly distributed in the culture dish.

[0082] (2) Prepare solution A and solution B

[0083] Solution A: 6.25ml 2×HEPES buffer (the amount packed in 5 large dishes, the effect is the best).

[0084] Solution B: a mixture obtained by adding the following plasmids: 112.5 μg PRRLSIN-NKG2D (targetplasmid); 39.5 μg pMD2.G (VSV-G envelope); 73 μg pCMVR8.74 (gag, pol, tat, rev); 625 μl 2M calcium ion solution. Total volume of solution A: 6.25ml.

[0085] Mix solution B well, and while vortexing solution A gently, add solution B drop by drop to obtain a mixed solution of A and B, and let stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, and add it dropwise to the culture dish co...

Embodiment 3

[0086] Example 3 Preparation of NKG2D CAR-NK cells

[0087] Adjust the NK-92 cell density to 2~3×10 5 / ml, add lentivirus (prepared in Example 2) according to the volume ratio (V / V) lentivirus: cell culture medium = 1:5, and add polybrene 8 μg / ml at the same time. After 4 h, add an equal amount of fresh complete medium to adjust the NK-92 cell density to 1×10 5 / ml to continue the culture. The next day, all the cells were centrifuged, fresh medium was added, and the culture was continued. Replenish fluid every 1-2 days to maintain cell density at 2-3×10 5 / ml. After 72 hours, CAR antibody staining was performed, and NKG2D CAR NK-92 positive cells were sorted by flow cytometry and expanded for culture. Observe the color change of the medium, cell density, and cell shape every day and make corresponding records.

[0088] The positive rate of NKG2D CAR NK-92 cells was detected by flow cytometry, and the results of flow cytometry were as follows: Figure 2a and Figure 2b...

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Abstract

The invention provides a chimeric antigen receptor, a NKG2D CAR-NK cell expressing the chimeric antigen receptor, a preparation method and an application thereof. The chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain, and the antigen binding domain is capable of specifically binding to a tumor specific antigen NKG2D ligand and activating NK cells through the transmembrane domain and the costimulatory signaling domain. The CAR-NK cell uses the NKG2D ligand as a target antigen and specifically uses the NKG2D CAR-NK cells to kill tumor cells. The cell can be used as a therapeutic drug for tumor diseases for the treatment of tumors with high expression of the ligand of NKG2D molecules, and provides a novel method for tumor prevention and treatment.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a chimeric antigen receptor, NKG2D CAR-NK cells expressing the chimeric antigen receptor, and a preparation method and application thereof. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) modified immune cells use genetic engineering to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize specific molecules on the tumor surface and the latter is used to initiate an immune cell response after recognizing tumor surface molecules and exert cytotoxicity. It mainly uses T-cells as carriers, but when CAR-T cells treat tumors, cytokines such as IL-6 will increase sharply, resulting in cytokine storm phenomenon, and there are problems such as on-target / off-target toxicity and neurotoxicity, and in severe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
CPCC07K16/2833C07K14/70517C07K14/70578C07K14/7051C12N5/0646C12N15/86A61P35/00C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N2510/00C12N2800/107C12N2740/15043A61K39/464429A61K2239/38A61K39/4631A61K39/4613C12N15/62C12N15/867C12N15/63A61K2039/812A61K2039/86C12N2501/515C12N2501/505A61K9/0019C07K16/3015C07K16/3023C07K16/3046
Inventor 韩昆昆韩俊峰李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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