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Method for preparing CAR-T cell from gamma delta T cell derived from umbilical cord blood, CAR-T cell and application

A cord blood source and cell technology, applied in the direction of cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, genetically modified cells, etc., can solve off-target effects, insertion mutations, solid tumors No significant curative effect and other problems have been achieved, and the effect of good safety and elimination of rejection is achieved

Pending Publication Date: 2019-04-12
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CAR-T cell immunotherapy has some areas that need to be improved, such as off-target effects, cytokine storm, insertion mutation, etc., and has not yet achieved significant curative effect on solid tumors. It is important to develop new effector cells with strong anti-tumor effects. Theoretical significance and clinical application value

Method used

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  • Method for preparing CAR-T cell from gamma delta T cell derived from umbilical cord blood, CAR-T cell and application
  • Method for preparing CAR-T cell from gamma delta T cell derived from umbilical cord blood, CAR-T cell and application
  • Method for preparing CAR-T cell from gamma delta T cell derived from umbilical cord blood, CAR-T cell and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 Construction of recombinant lentiviral vector BRD-PTK--mCD19-01 (see figure 1 )

[0044] Using the plasmids PLVX-EF1α-CD19 and plasmid pLVX-EF1α-IRES-Puro preserved in the R&D Department as templates, primers were designed to PCR amplify fragments CD19-CD3ξ, RRE-Cppt / CTS-EF1α, and Overlap PCR was used to amplify to obtain enzyme-containing Cut site NotI-HF and BamHI-HF fragment RRE-Cppt / CTS-EF1α-CD19-CD3ξ;

[0045] The correctly constructed third-generation lentiviral backbone plasmid PTK881-CD33 was double-digested with NotI-HF and BamHI-HF restriction endonucleases, and the product was subjected to 0.8% agarose gel electrophoresis, and the gel was recovered and placed in an Eppendorf tube. Agarose Gel Recovery Kit recovers the corresponding fragments, and determines the purity and concentration of the product.

[0046] Add the fragments to an Eppendorf tube at a molar ratio of 1:2, add ExnaseⅡligase and homologous recombinase 5×CEⅡbuffer, and react at 3...

Embodiment 2B

[0048] Preparation of embodiment 2BRD-PTK-mCD19-01 plasmid (see figure 2 )

[0049] 1. Plasmid Preparation

[0050] Inoculate the DH5alpha strain containing the plasmid BRD-PTK-mCD19-01 into 250 mL of LB culture solution containing 100 μg / mL ampicillin, and culture overnight at 37°C and 190 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0051] Take out the Buffers P1 in the plasmid extraction kit, add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, and shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1.

[0052] Add 120mL Buffers P2 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to 50rpm, mix thoroughly and place at room temperature for 5min.

[0053] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of t...

Embodiment 3

[0074] Example 3 Preparation of Lenti3-mCD19CAR lentivirus

[0075] 1. Lentivirus preparation

[0076] Insert 130.0-140.0×10 in the multilayer cell culture flask 6 Number of 293T cells, a total of 560mL DMEM complete medium, cultured in a 37°C 5% CO2 incubator for 24 hours. Add DMEM complete medium mixed with 320 μg of plasmid (mass ratio: PTK-mCD19:pMDLg:pRSV:pMD2.G=12:10:5:6) into a tube containing 960 μg of PEI, vortex, and mix thoroughly. The volume of the mixed solution was 35 mL, and it was equilibrated at room temperature for 10 min. Mix the above-mentioned 35mL PEI-plasmid mixture with 525mL DMEM complete medium, and replace it into the above-mentioned multi-layer cell culture flask. After the multi-layered cell culture flask was placed in an incubator containing 5% (v / v) CO2 at 37° C. for 3 days, the cell culture supernatant was collected.

[0077]After the supernatant was centrifuged at 4000rpm (or 3000g) for 30min, the lentiviral supernatant was suction-filtered...

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Abstract

The invention discloses a method for preparing a CAR-T cell from gamma delta T cell derived from umbilical cord blood, the CAR-T cell and an application and also relates to nucleic acid coding a chimeric antigen receptor and a recombinant expression vector expressing the chimeric antigen receptor. The umbilical cord blood is used as a source of the gamma delta T cell, the gamma delta T cell is separated from the umbilical cord blood, after efficient amplification, the T cell is transduced by a lentiviral vector loaded with the chimeric antigen receptor, and the CAR-T cell can be obtained, so that positive tumor cells or other infectious cells which can efficiently or specifically kill related antigens are obtained. The CAR-T cell will play roles in tumor cell therapy and immunotherapy.

Description

technical field [0001] The invention belongs to the field of cell therapy and biopharmaceuticals; in particular, it relates to a method for preparing CAR-T cells from cord blood-derived γδT cells, the CAR-T cells and applications. Background technique [0002] The full name of CAR-T is Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric Antigen Receptor T-cell Immunotherapy, which is considered to be one of the most promising tumor treatment methods, bringing new hope to cancer patients. However, CAR-T cell immunotherapy has some areas that need to be improved, such as off-target effects, cytokine storm, insertion mutation, etc., and has not yet achieved significant curative effect on solid tumors. It is important to develop new effector cells with strong anti-tumor effects. Theoretical significance and clinical application value. [0003] Human T lymphocytes can be divided into αβ and γδT cells according to the structure of their cell surface receptors (TCR). group. ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/30C07K2319/02C07K2319/33C12N15/86C12N2510/00C12N2740/15043
Inventor 张同存顾潮江
Owner WUHAN BIO RAID BIOTECH CO LTD
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