Chimeric antigen receptor and preparation method thereof, NK cell modified by chimeric antigen receptor and application in treating HBV infection

A technology of chimeric antigen receptors and NK cells, applied in the field of genetic engineering, can solve problems such as inability to identify subtypes and limit the scope of treatment, and achieve the effect of reducing therapeutic doses and side effects

Active Publication Date: 2019-01-25
上海兴瑞一达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, less than 10% of patients treated with interferon produced a sustained therapeutic effect
[0005] Due to the many subtypes of HBV viru

Method used

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  • Chimeric antigen receptor and preparation method thereof, NK cell modified by chimeric antigen receptor and application in treating HBV infection
  • Chimeric antigen receptor and preparation method thereof, NK cell modified by chimeric antigen receptor and application in treating HBV infection
  • Chimeric antigen receptor and preparation method thereof, NK cell modified by chimeric antigen receptor and application in treating HBV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Chimeric antigen receptors, such as figure 1 As shown, it includes the artificial nucleic acid sequence of leader, the artificial nucleic acid sequence of HBsAg single-chain antibody, the artificial nucleic acid sequence of CD8α hinge region, the artificial nucleic acid sequence of NKG2D transmembrane region, the artificial nucleic acid sequence of 2B4 co-stimulatory region, the artificial nucleic acid sequence of CD3ζ signal transduction region, and the artificial sequence of T2A autonomous region. The nucleic acid artificial sequence of the cutting region, the artificial nucleic acid sequence of IFN-α, and the artificial nucleic acid sequence of the RQR8 molecular switch region.

[0051] Wherein, the RQR8 nucleic acid sequence has the nucleic acid sequence as described in SEQ ID NO.10. RQR8 is composed of signal peptide nucleic acid sequence, CD20 mimic epitope nucleic acid sequence, CD34 mimic epitope nucleic acid sequence, CD20 mimic epitope nucleic acid sequence, C...

Embodiment 2

[0055] A method for preparing a chimeric antigen receptor, comprising the steps of:

[0056] (1) Artificial nucleic acid sequence of leader, artificial nucleic acid sequence of HBsAg single-chain antibody, artificial nucleic acid sequence of CD8 α hinge region, artificial nucleic acid sequence of NKG2D transmembrane region, artificial nucleic acid sequence of 2B4 co-stimulatory region, artificial nucleic acid sequence of CD3ζ signal transduction region, T2A self-slicing region nucleic acid artificial sequence, IFN-α nucleic acid artificial sequence, T2A self-slicing region nucleic acid artificial sequence, RQR8 molecular switch region nucleic acid artificial sequence synthesize the entire expression frame and insert it into the standard vector pUC to obtain pUC-Anti-HBsAg -IFN-α-CAR;

[0057] (2) Carry out double digestion of pUC-Anti-HBsAg-IFN-α-CAR, use agar gel electrophoresis to cut out the agar part of the DNA fragment of Anti-HBsAg-IFN-α-CAR, use the DNA extraction kit s...

Embodiment 3

[0063] Prepare a plasmid containing the Anti-HBsAg-IFN-α-CAR DNA fragment, and ligate the purified Anti-HBsAg-IFN-α-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment overnight at 16°C to form pLent- Anti-HBsAg-IFN-α-CAR plasmid, comprising the following steps:

[0064] According to artificial nucleic acid sequence of Leader, artificial nucleic acid sequence of HBsAg single-chain antibody, artificial nucleic acid sequence of CD8 hinge region, artificial nucleic acid sequence of NKG2D transmembrane region, artificial nucleic acid sequence of 2B4 co-stimulatory region, artificial nucleic acid sequence of CD3 ζ signal transduction region, T2A self-shearing region The nucleic acid artificial sequence of the cutting region, the artificial nucleic acid sequence of the IFN-α molecular switch region, the artificial nucleic acid sequence of the T2A self-cleaving region, and the artificial nucleic acid sequence of the RQR8 molecular switch region entrust Sangon Bioengineering (...

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Abstract

The invention relates to a chimeric antigen receptor and a preparation method thereof, an NK cell modified by the chimeric antigen receptor and application in treating HBV infection. The chimeric antigen receptor comprises a leader nucleotide artificial sequence, an HBsAg single-chain antibody nucleotide artificial sequence, a CD8alpha hinge region nucleotide artificial sequence, an NKG2D transmembrane region nucleotide artificial sequence, a 2B4 co-stimulation region nucleotide artificial sequence, a CD3zeta signal transduction region nucleotide artificial sequence, a T2A self-cleavable region nucleotide artificial sequence, an IFN-alpha nucleotide artificial sequence and a RQR8 molecular switch region nucleotide artificial sequence. According to the chimeric antigen receptor and the preparation method thereof, the NK cell modified by the chimeric antigen receptor and the application in treating HBV infection, for a common alpha determinant specific antibody, HBsAg in different subtypes can be identified, therefore, the Anti-HBsAg-IFN-alpha-CARNK cell has therapeutic effects on all HBV subtype infection, and meanwhile, the therapeutic dosage and toxic and side effects are also reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a chimeric antigen receptor, a preparation method thereof, an NK cell modified by using the same and an application for treating HBV infection. Background technique [0002] HBV infection is a global public health problem that threatens human health. It is an infectious disease caused by hepatitis B virus (Hepatitis B virus, HBV). It is estimated that about 2 billion people in the world have ever been infected with hepatitis B virus, of which chronic HBV infection has reached nearly 240 million to 350 million, and 25% of them have liver cirrhosis or cancer due to lifelong infection caused by HBV. Currently recognized chemotherapy methods include interferon-α (Interferon-α, IFN-α) and nucleoside analogs. Nucleoside analogs block the synthesis of viral DNA by multifunctional reverse transcriptase. Oral nucleotide analogs can effectively reduce the level of HBV-DNA in ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/867A61K35/17A61P31/20
CPCA61K35/17A61P31/20C07K14/7051C07K16/082C07K2317/76C07K2319/33C12N15/86C12N2740/15043
Inventor 刘明录王立新韩国英金海锋张传鹏冯建海韩庆梅刘敏王亮万磊
Owner 上海兴瑞一达生物科技有限公司
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