NKG2D-CAR-T (Chimeric Antigen Receptor-T) cell of co-expression cell factor IL-7 and application thereof

A co-expression and host cell technology, applied in the field of biomedicine, can solve problems such as deletion

Active Publication Date: 2019-02-05
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the remarkable clinical response achieved by CAR-T cell immunotherapy for hematological malignancies, the use of CAR-T cells to treat solid tumors is limited by the tissue structure of solid tumors and a strong immunosuppressive environment. Another key deficiency of

Method used

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  • NKG2D-CAR-T (Chimeric Antigen Receptor-T) cell of co-expression cell factor IL-7 and application thereof
  • NKG2D-CAR-T (Chimeric Antigen Receptor-T) cell of co-expression cell factor IL-7 and application thereof
  • NKG2D-CAR-T (Chimeric Antigen Receptor-T) cell of co-expression cell factor IL-7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0205] Example 1 Acquisition of NKG2D extracellular segment gene sequence and human IL-7 gene full-length sequence

[0206]Check the CDS region of the full-length NKG2D gene through the website https: / / www.ncbi.nlm.nih.gov / pubmed / , and find the extracellular region of the NKG2D protein at https: / / www.uniprot.org / , Find the corresponding extracellular segment gene sequence in the NKG2D full-length gene sequence. Since NKG2D protein is a type II transmembrane protein, its extracellular segment sequence is located at the N-terminal and does not contain a signal peptide. Therefore, we added the signal peptide sequence in the existing CD19-CAR to the front end of the NKG2D extracellular segment sequence. Use SnapGene software to design and synthesize primers (the primers contain cell peptide sequences) in the company, use human T cell cDNA as a template, amplify the extracellular segment of NKG2D by RT-PCR, and sequence the RT-PCR products to obtain Sig- The sequence of the extrac...

Embodiment 2

[0214] Example 2 Construction of PCDH-NKG2D-CAR and PCDH-NKG2DIL7-CAR vectors

[0215] The obtained sigNKG2DEX sequence and the already constructed second-generation CAR sequence (CD8-CD3zeta-4-1BB) (obtained from Shanghai Bangyao Biotechnology Co., Ltd.) were connected together by overlapping PCR. After RT-PCR and sequencing identification, it was confirmed that the construction was successful, and the PCDH-NKG2D-CAR vector was obtained. On the basis of this vector, the IL-7 gene sequence was constructed between CAR and GFP by overlapping PCR, and after RT-PCR and sequencing identification. It is confirmed that the construction is successful, and the PCDH-NKG2DIL7-CAR vector is obtained, and the structure diagram is as follows figure 1 shown.

Embodiment 3

[0216] Embodiment 3 virus packaging

[0217] Amplification and viral packaging of PCDH-NKG2D-CAR and PCDH-NKG2DIL7-CAR plasmids

[0218] 3.1 Plasmid transfection

[0219] Put the plasmid, PEI, and Opti-MEM medium at room temperature for 5 minutes;

[0220] 2) Take 436 μl of Opti-MEM into a 1.5ml EP tube, then add 64 μg PEI, mix well, and let stand at room temperature for 5 minutes;

[0221] 3) Take 12 μg of vector plasmids PCDH-NKG2D-CAR and PCDH-NKG2DIL7-CAR, 8 μg of psPA×2, 4 μg of pMD2.G, add Opti-MEM to 500 μl, and let stand at room temperature for 5 minutes;

[0222] 4) Add the prepared PEI-Opti-MEM solution into the Opti-MEM containing the plasmid, and let stand at room temperature for 20 minutes;

[0223] 5) Slowly drop 1ml of the DNA / PEI mixture into the 293T Petri dish laid out the day before, mix gently, incubate in a 37°C incubator, replace with fresh medium after 6-8h, and put it in a 37°C incubator to continue incubating .

[0224] 3.2 Virus collection and co...

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Abstract

The invention provides an NKG2D-CAR-T (Chimeric Antigen Receptor-T) cell of a co-expression cell factor IL-7 and application thereof to tumor killing, and particularly provides a CAR of a specific targeted NKG2D ligand protein and a CAR-T cell thereof. The CAR comprises an antigen binding domain and/or IL-7. The CAR-T cell provided by the invention has a good tumor killing effect.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to NKG2D-CAR-T cells co-expressing cytokine IL-7 and its application in tumor killing. Background technique [0002] Tumor is a disease that seriously threatens human life. With the development of medicine and science, many treatment options for tumors have been born, such as surgical resection, chemical drug therapy and radiation therapy. It has a good curative effect and can significantly alleviate the symptoms of tumor patients and prolong the survival period of patients. However, for patients with relapsed and refractory tumors and patients with tumor metastasis, the above methods often have little effect. In recent years, a new tumor immunotherapy therapy has gradually come into people's field of vision, namely chimeric antigen receptor T cell therapy (CAR-T). Some in-depth studies of preclinical and clinical trials have shown that CAR-T therapy has achieved encouraging ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/12A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/5418C07K14/7051C07K16/18C07K2319/03
Inventor 江文正何聪王熙刘明耀席在喜
Owner EAST CHINA NORMAL UNIV
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