Universal chimeric antigen receptor T cell targeting claudin 18.2, construction method and application of universal chimeric antigen receptor T cell

A chimeric antigen receptor and targeting technology, applied in the field of molecular biomedicine, can solve problems such as unknown functions, and achieve the effect of simple structure and high tumor killing activity

Inactive Publication Date: 2020-12-22
NANJING BIOHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Claudin family proteins were discovered less than 20 years ago, and now 27 genes are known, but most of them have unknown functions

Method used

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  • Universal chimeric antigen receptor T cell targeting claudin 18.2, construction method and application of universal chimeric antigen receptor T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of lentiviral expression vector

[0034] A nucleic acid molecule encoding the signal transduction region was synthesized, the nucleotide sequence of which was shown in SEQ ID NO.5, and named pGSI.

[0035] 3ug pGSI and recombinant lentiviral vector plasmid pCDH-EF1 were double-digested with BamHI and SalI restriction endonucleases respectively, and the digested products were recovered from the gel and ligated with T4 DNA ligase, ligated overnight at 4°C, transformed into DH5α competent cells, and taken Spread 100 μL of the bacterial solution onto an LB plate containing ampicillin resistance, and incubate overnight at 37°C. Single clones were picked for colony PCR, positive clones were sent for sequencing, and the clones with correct sequencing results were saved and plasmids were extracted, named pCDH-EF1-emCAR.

[0036] A nucleic acid molecule encoding the antigen-binding region was synthesized, the nucleotide sequence of which was shown in SEQ ...

Embodiment 2

[0038] Example 2 Lentiviral packaging

[0039] Take out a tube of frozen 293T cells from liquid nitrogen and quickly place them in a 37°C water bath until the ice cubes disappear, add dropwise to a 15ml centrifuge tube containing 5ml preheated medium, centrifuge at 1200rpm for 3min, discard the supernatant, and culture with 293T Medium (10% FBS+DMEM) resuspended cells were inoculated into 150mm culture dishes, 37°C, 5% CO 2 Cultivate in saturated humidity. When the confluence of the cells reaches more than 90%, discard the old medium, add 5ml sterilized PBS solution, shake gently, discard the PBS solution after washing the cells, add 2ml 0.25% Trypsin-EDTA digestion solution, and digest for 1-2min until Cells are fully digested. Add serum-containing medium to stop the digestion, centrifuge the cell suspension at 1200rpm for 3min, resuspend the centrifuged cells with medium, inoculate 1.2×107 cells in each 150mm culture dish for packaging lentivirus, 37°C, 5% CO 2 Culture in...

Embodiment 3

[0042] Example 3 Lentivirus purification

[0043] At 24h and 48h after the medium exchange, the supernatant was collected and temporarily stored at 4°C, and 20ml of fresh medium was replaced. Centrifuge the collected liquid at 4°C and 3500rpm for 15min, discard the precipitate, concentrate the supernatant with a Millipore protein ultrafiltration column (10KD) and measure the virus titer at the same time, dilute the lentivirus claudin CAR) to 1*10 8 TU / ml, the aliquoted lentivirus (claudin CAR) was stored at -80°C.

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Abstract

The invention relates to a universal chimeric antigen receptor T cell targeting claudin 18.2, a construction method and an application of the universal chimeric antigen receptor T cell, and belongs tothe technical field of molecular biomedicine. The invention provides a single-chain antibody targeting the claudin 18.2, and the chimeric antigen receptor T cell containing the single-chain antibodytargeting the claudin 18.2. The chimeric antigen receptor targeting the claudin 18.2 can specifically target the claudin 18.2, promote the amplification of the T cell in the body of a patient, efficiently and specifically kill tumor cells, and hardly cause damage to normal cells.

Description

technical field [0001] The invention relates to a general-purpose chimeric antigen receptor T cell targeting claudin 18.2, a construction method and application thereof, and belongs to the technical field of molecular biomedicine. Background technique [0002] Gastric cancer is the malignant tumor with the third fatality rate in the world. According to the data of the World Health Organization in 2015, 754,000 gastric cancer patients die every year in the world; more than 600,000 new cases of gastric cancer occur in my country every year, ranking second in the incidence of malignant tumors. endanger human life and health. The curative effect of existing treatment methods for advanced gastric cancer is still unsatisfactory, and the five-year survival rate is only 5-20%. Therefore, it is urgent to seek safe and effective treatment methods. [0003] Chimeric antigen receptor T-cell immunotherapy (Chimeric antigen receptor T-cellimmunotherapy), referred to as CAR-T therapy, is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61K39/0011A61K2039/5156A61K2039/5158A61P35/00A61K2039/828A61K2039/852C07K16/28C07K2317/622C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 贺小宏王延宾任江涛
Owner NANJING BIOHENG BIOTECH CO LTD
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