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Anti-egfrvⅢ safe chimeric antigen receptor, its preparation method, nk cell modified by using it and application

A chimeric antigen receptor, NK cell technology, applied in the field of genes, to achieve the effect of small molecular weight, avoid toxic side effects, and efficiently and quickly induce apoptosis

Active Publication Date: 2021-05-25
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in order to finally overcome this cancer, it is necessary to solve the immunosuppressive effect in the tumor microenvironment, the safety of CAR-T, and the industrialization of CAR-T.

Method used

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  • Anti-egfrvⅢ safe chimeric antigen receptor, its preparation method, nk cell modified by using it and application
  • Anti-egfrvⅢ safe chimeric antigen receptor, its preparation method, nk cell modified by using it and application
  • Anti-egfrvⅢ safe chimeric antigen receptor, its preparation method, nk cell modified by using it and application

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Experimental program
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Effect test

Embodiment 1

[0042] Anti-EGFRvIII safe chimeric antigen receptor, such as figure 1As shown, its nucleic acid sequence must contain a molecular switch RQR8 nucleic acid sequence, and the RQR8 nucleic acid sequence has a nucleic acid sequence as described in SEQ ID NO.9. RQR8 is composed of signal peptide nucleic acid sequence (SEQ ID NO.10), CD20 mimic epitope nucleic acid sequence (SEQ ID NO.11), CD34 mimic epitope nucleic acid sequence (SEQ ID NO.12), CD20 mimic epitope The nucleic acid sequence (SEQ ID NO.11) and the CD8 hinge transmembrane region nucleic acid sequence (SEQ ID NO.13) are sequentially concatenated, and the complete nucleic acid sequence is shown in SEQ ID NO.9. Sangon Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize the nucleotide sequence.

[0043] In this embodiment, the anti-EGFRvIII safe chimeric antigen receptor comprises the CD8leader nucleic acid artificial sequence as described in SEQ ID NO.2; the EGFRvIII scFv nucleic acid artificial sequence a...

Embodiment 2

[0046] A method for preparing an anti-EGFRvⅢ safe chimeric antigen receptor, comprising the steps of:

[0047] (1) Synthesize the entire expression cassette according to the CD8leader nucleic acid sequence, EGFRvIII scFv nucleic acid sequence, CD8α nucleic acid sequence, NKG2D nucleic acid sequence, 2B4 nucleic acid sequence, CD3ζ nucleic acid sequence, self-cleaving polypeptide T2A nucleic acid sequence and RQR8 nucleic acid sequence and insert it into the standard vector pUC Above, get pUC-Anti-EGFRvIII CAR;

[0048] (2) Carry out double digestion of pUC-Anti-EGFRvIII CAR, use agar electrophoresis to cut out the agar part of the Anti-EGFRvIII CAR DNA fragment, use the DNA extraction kit sol solution to treat, pass through the DF column and discard the filtrate, rinse the DF column, empty Centrifuge and elute the DF column, collect the centrifugate, and obtain the purified Anti-EGFRvIII CAR DNA fragment, which is the anti-EGFRvIII safe chimeric antigen receptor.

[0049] In ...

Embodiment 3

[0055] To prepare a plasmid containing the Anti-EGFRvIII CAR DNA fragment, the purified Anti-EGFRvIII CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were ligated overnight at 16°C to form a pLent-Anti-EGFRvIIICAR plasmid, including the following steps:

[0056] According to the guide CD8leader nucleic acid artificial sequence, EGFRvIII scFv nucleic acid artificial sequence, CD8α nucleic acid artificial sequence, NKG2D nucleic acid artificial sequence, 2B4 nucleic acid artificial sequence, CD3ζ nucleic acid artificial sequence, self-cleaving polypeptide T2A nucleic acid artificial sequence, RQR8 nucleic acid artificial sequence, entrusted Sangon Bioengineering (Shanghai) Co., Ltd. synthesized the entire expression cassette and inserted it into the standard vector pUC, so it was named pUC-Anti-EGFRvIII CAR,

[0057] At the same time, the pUC-Anti-EGFRvIII CAR and pLent-C-GFP vectors were digested with Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest N...

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Abstract

The invention belongs to the anti-EGFRvIII safe chimeric antigen receptor, its preparation method, NK cells modified by using it and its application. The anti-EGFRvIII safe chimeric antigen receptor comprises CD8leader nucleic acid sequence, EGFRvIII scFv nucleic acid sequence and CD8α nucleic acid sequence , NKG2D nucleic acid sequence, 2B4 nucleic acid sequence, CD3ζ nucleic acid sequence, self-cleaving polypeptide T2A nucleic acid sequence and RQR8 nucleic acid sequence. The anti-EGFRvIII safe chimeric antigen receptor provided by the present invention can reduce severe toxic side effects caused by excessive activity or uncontrolled activity of the chimeric antigen receptor after it is injected into the body.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to an anti-EGFRvIII safe chimeric antigen receptor, a preparation method thereof, NK cells modified by the same and application thereof. Background technique [0002] Glioblastoma (GBM) is currently the most common malignant tumor of the central nervous system, and the overall median survival time of patients is only about 15 months. Current treatment options for the disease include surgery, radiation, and chemotherapy, but the tumor often becomes resistant to treatment and recurs. [0003] Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific antigen with a high expression rate in malignant tumors such as glioblastoma, and is closely related to the occurrence and development of tumors. Molecular targets ideal for targeted therapy. Current clinical trials have shown that targeting EGFRvⅢ chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/62C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2863C07K2319/33C12N15/86C12N2510/00C12N2740/15043
Inventor 刘明录王立新韩国英万磊刘敏强邦明王亮
Owner SHANDONG XINRUI BIOTECH CO LTD
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