Preparation method and application of CAR-T cell taking HIV-1 gp120 and CD20 as double targets

A 3BNC117-CD20CAR, hiv-1gp120 technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve problems that have not been reported and achieve good application prospects

Pending Publication Date: 2022-05-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the dual-target simultaneously targeting HIV-1gp120 and CD20

Method used

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  • Preparation method and application of CAR-T cell taking HIV-1 gp120 and CD20 as double targets
  • Preparation method and application of CAR-T cell taking HIV-1 gp120 and CD20 as double targets
  • Preparation method and application of CAR-T cell taking HIV-1 gp120 and CD20 as double targets

Examples

Experimental program
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Effect test

Embodiment 1

[0109] Example 1 Construction of a chimeric antigen receptor expression vector containing 3BNC117 and CD20 dual targets in vitro

[0110] Firstly, using the pCDH-CMV-MCS-EF1α-Puro plasmid as the backbone, the MCS region was cleaved with EcoRI and BamHI endonucleases. Then, using the pTRPE-3BNC117-G4H-BBz plasmid as a template, the 3BNC117 CAR fragment was amplified by PCR using onestep-3BNC117-DNR-1F and onestep-3BNC117-DNR-1R primers. Finally, the digestion product and the PCR product were gel recovered and then connected by Onestep homologous recombination. The ligation product was transformed in DH5α competent and then coated on Amp+ plate to screen positive clones. After the positive clones were expanded and cultured, plasmids were extracted and sequenced for verification. Positive plasmid pCDH-CMV-3BNC117-EF1α.

[0111] Then, using the constructed pCDH-CMV-3BNC117-EF1α plasmid as a template, the Puro fragment was excised with XmaI and SalI endonucleases. Using the CD20-pU...

Embodiment 23

[0112] Example 23B Preparation and in vitro functional verification of CD20 CAR-T cells

[0113] In order to obtain lentiviral particles expressing 3B-CD20 CAR, the present invention co-transfected the lentiviral backbone plasmid, △8.91 and VSVG into 293T cells, collected the virus supernatant after 48 hours, filtered, concentrated by ultracentrifugation and placed in - Store at 80°C for later use.

[0114] Infection of healthy human CD3 by lentivirus carrying the 3B-CD20 CAR element + T lymphocytes prepare effector cells. After obtaining the peripheral blood of healthy donors, the PBMCs cells were separated by the lymphocyte separation medium, and then the human CD3 cells were obtained by magnetic bead sorting. + T lymphocytes. Then CD3 / 28 activated magnetic beads were added at a ratio of magnetic beads: cells = 1:1. 24h later, the lentivirus expressing 3B-CD20 CAR was used to infect CD3 with MOI=20 static infection + T cells prepared dual-target CAR-T effector cells and...

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Abstract

The invention belongs to the technical field of biomedicine, and relates to an infectious disease and tumor complication immune technology, in particular to a preparation method and application of a CAR-T cell with HIV-1 gp120 and CD20 as double targets. The invention also provides a lentiviral vector based on the combination of a chimeric antigen receptor of a widely neutralizing antibody 3BNC117 and an anti-CD20 chimeric antigen receptor. The chimeric antigen receptor disclosed by the invention comprises a single-chain antibody ScFv targeting HIV-1 gp120, an IgG4 hinge region, a CD8 transmembrane region, 4-1BB and a zeta chain of a leukocyte antigen differentiation group 3. Tests show that the double-target CAR-T cell can efficiently and specifically remove HIV infected cells and lymphoma cells, is used for preparing drugs and reagents for treating AIDS-related lymphoma, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a preparation method of a CAR-T cell with HIV-1 envelope protein gp120 and CD20 as dual targets and its application in the preparation of a live cell drug against AIDS-related lymphoma. Background technique [0002] According to reports, AIDS has become a serious public health and social problem in the world today. Antiretroviral therapy (ART) currently used clinically can effectively reduce the plasma viral load of patients, but cannot cure AIDS. At the same time, AIDS-related lymphoma still accounts for more than 50% of HIV-related malignancies and is the leading cause of AIDS-related death. Therefore, there is an urgent need to find a new therapy to cure both AIDS and AIDS-related lymphoma. [0003] Chimeric antigen receptor T cell therapy (chemical antigen receptor T cell therapy, CAR-Ttherapy) was first carried out in clinical trials for the treatment of HIV-1 patients in the 1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K39/42A61P35/00A61P31/18
CPCC07K16/1063C07K16/2887C07K14/7051C07K14/70517C07K14/70578C12N15/86C12N5/0636A61K39/001124A61P35/00A61P31/18C07K2317/622C07K2317/76C07K2317/53C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/804A61K2039/505
Inventor 朱焕章姜正涛
Owner FUDAN UNIV
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