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Nucleic acid for treating HCC (Hepatocellular Carcinoma), preparation method thereof, CAR-T (Chimeric Antigen Receptor T) cell having nucleic acid and preparation method of CAR-T cell

A nucleic acid and cell technology, applied in the field of genes, can solve the problem that the tissue killing of liver cancer needs to be improved

Inactive Publication Date: 2018-11-30
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this CAR-T cell only has a killing rate for hepatocellular carcinoma cell lines in vitro, and the killing rate for liver cancer tissues in vivo still needs to be improved, and those skilled in the art have not made greater improvements

Method used

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  • Nucleic acid for treating HCC (Hepatocellular Carcinoma), preparation method thereof, CAR-T (Chimeric Antigen Receptor T) cell having nucleic acid and preparation method of CAR-T cell
  • Nucleic acid for treating HCC (Hepatocellular Carcinoma), preparation method thereof, CAR-T (Chimeric Antigen Receptor T) cell having nucleic acid and preparation method of CAR-T cell
  • Nucleic acid for treating HCC (Hepatocellular Carcinoma), preparation method thereof, CAR-T (Chimeric Antigen Receptor T) cell having nucleic acid and preparation method of CAR-T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The nucleic acid used for treating HCC includes at least the CD226 costimulatory region nucleic acid sequence as described in SEQ ID NO.6 and the 4-1BB costimulatory region nucleic acid sequence as described in SEQ ID NO.7.

[0044] In this example, if figure 1 Shown, the nucleic acid sequence that is used for the treatment of HCC comprises the CD8Leader nucleic acid sequence as described in SEQ ID NO.2 of sequential connection; The GPC3scFv nucleic acid sequence as described in SEQ ID NO.3; CD8α as described in SEQ ID NO.4 The nucleic acid sequence of the hinge region; the CD8α transmembrane region nucleic acid sequence as described in SEQ ID NO.5; the CD226 co-stimulatory region nucleic acid sequence as described in SEQ ID NO.6; the 4-1BB co-stimulatory region as described in SEQ ID NO.7 Stimulatory region nucleic acid sequence; CD3ζ signaling region nucleic acid sequence as described in SEQ ID NO.8.

Embodiment 2

[0046] A method for preparing a nucleic acid for treating HCC, comprising the steps of:

[0047] (1) According to artificial nucleic acid sequence of Leader, artificial nucleic acid sequence of GPC3 single-chain antibody, artificial nucleic acid sequence of CD8 hinge region, artificial nucleic acid sequence of CD8 transmembrane region, artificial nucleic acid sequence of CD226 costimulatory region, artificial nucleic acid sequence of 4-1BB costimulatory region , CD3ζ signaling region nucleic acid artificial sequence to synthesize the entire expression frame and insert it into the standard vector pUC to obtain pUC-GPC3-226BB-CAR;

[0048] (2) Carry out double enzyme digestion of pUC-GPC3-226BB-CAR, use agar electrophoresis to cut off the agar part of the GPC3-226BB-CAR DNA fragment agar, use the DNA extraction kit sol solution to treat, pass through the DF column, discard the filtrate, and rinse the DF Column, empty, elute the DF column, collect the centrifuge, and obtain the p...

Embodiment 3

[0053] A plasmid, the plasmid is the pLent-GPC3-226BB-CAR plasmid obtained by inserting the fusion gene fragment GPC3-226BB-CAR DNA into the lentiviral expression vector pLent-C-GFP. The plasmid was prepared by the following preparation method: the above-mentioned purified GPC3-226BB-CAR DNA fragment and the linearized pLent-C-GFP DNA fragment were combined in the ligation system: 10×buffer: 1 μl; T4 ligase: 1 μl; GPC3 -226BB-CAR DNA fragment: 4 μl; linearized pLent-C-GFP DNA fragment: 4 μl, ligation formed.

[0054] In this example, the plasmid was prepared by the following method:

[0055] According to artificial sequence of Leader nucleic acid, artificial sequence of GPC3 single-chain antibody nucleic acid, artificial sequence of nucleic acid of CD8 hinge region, artificial sequence of nucleic acid of CD8 transmembrane region, artificial sequence of nucleic acid of CD226 costimulatory region, artificial sequence of nucleic acid of 4-1BB costimulatory region, CD3ζ signal Th...

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Abstract

The invention discloses nucleic acid for treating HCC (Hepatocellular Carcinoma), a preparation method thereof, a CAR-T (Chimeric Antigen Receptor T) cell having the nucleic acid and a preparation method of the CAR-T cell. A sequence of the nucleic acid at least comprises a CD226 costimulatory zone nucleic acid sequence as shown in SEQ ID NO.6 and a 4-1BB costimulatory zone nucleic acid sequence as shown in SEQ ID NO.7. An inhibition function of an HCC microenvironment can be overcome by promoting the generation of cell factors of IFN (Interferon)-gamma, so that the infiltration rate, the proliferation rate and the survival rate of T cells can be increased; after GPC3-226BB-CART cells are injected to a body, the GPC3-226BB-CART cells can survive in tumor and can be continuously proliferated until the tumor is removed.

Description

technical field [0001] The present invention relates to the field of gene technology, in particular to a nucleic acid for treating HCC, a preparation method thereof, CAR-T cells with the nucleic acid, and a preparation method of the cells. Background technique [0002] Hepatocellular carcinoma (HCC), referred to as liver cancer, is one of the most common malignant tumors in the world. Its incidence rate ranks fifth in the world and its mortality rate ranks second in the world. In February 2018, the National Cancer Center released the latest national cancer statistics. There are 854,000 new cases of primary liver cancer every year in the world, and 466,000 in China, accounting for about 55% of the world; deaths due to primary liver cancer every year 810,000 cases, 422,000 in China, accounting for about 45% -50% of the world. Current treatments for common early-stage liver cancer include surgery, liver transplantation, and radiation therapy. However, many patients are not su...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70503C07K14/7051C07K14/70517C07K14/70578C07K16/303C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 刘明录王立新韩国英韩庆梅金海锋张传鹏强邦明马洪华
Owner SHANDONG XINRUI BIOTECH CO LTD
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