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A bispecific chimeric antigen receptor, gene, construction method and application for treating HIV infection

A chimeric antigen receptor, bispecific technology, applied in the field of medicine and biology, can solve the problem that CAR-T cannot be effectively removed, and achieve the effect of huge clinical promotion value.

Active Publication Date: 2022-05-10
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since gpl20 and gp41 are not expressed or expressed very low in latent cells, CAR-T cannot be effectively cleared

Method used

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  • A bispecific chimeric antigen receptor, gene, construction method and application for treating HIV infection
  • A bispecific chimeric antigen receptor, gene, construction method and application for treating HIV infection
  • A bispecific chimeric antigen receptor, gene, construction method and application for treating HIV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of PTK881-EF1α-N6-MDE-8-01 plasmid

[0035] 1. The sequences SP1-N6 (nucleotide sequence SEQ ID NO.1) and MDE-8 (nucleotide sequence SEQ ID NO.2) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the synthesized sequences were cloned into the pUC57 vector superior.

[0036]2. Using the human cDNA library as a template, design primers to amplify fragments CD8 hinge, CD28 transmembrane region, 4-1BB, and CD3ζ respectively, and obtain short fragments of Strep tag II and G4S1 by primer complementation. Using Overlap PCR technology, SP1-scFv-N6, Strep tagⅡ, G4S1, MDE-8, CD8 hinge, CD28 transmembrane region, 4-1BB, CD3ζ were sequentially amplified and connected into EcoR I and BamH I with restriction sites The structure diagram of N6-MDE-8-CAR is as follows figure 1 shown.

[0037] Among them, N6-MDE-8-CAR is a single-chain antibody ScFv-N6 and ScFv-MDE-8 with a signal peptide that can recognize the surface of blood tumor cells infec...

Embodiment 2

[0042] Example 2: Preparation and sequencing of plasmids

[0043] 1. Preparation of plasmid

[0044] The DH5α strains containing the plasmid PTK881-EF1α-N6-MDE-8-1 were inoculated into 250 mL of LB culture medium containing 100 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0045] Take out Buffers P1 from the EndoFree plasma mega kit (Qiagen), add 120mL pre-cooled Buffers P1 to the centrifuged E. coli pellet, cover the cap of the centrifuge bottle, shake the centrifuge bottle vigorously to completely disperse the E. coli pellet in Buffers P1 .

[0046] 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.

[0047] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of the ...

Embodiment 3

[0060] Example 3, Preparation of Lenti3-N6-MDE-8-CAR Lentiviral Vector and Live Droplet Detection

[0061] 1. Preparation of lentiviral vector

[0062] Insert 130.0~140.0×10 in the multilayer cell culture flask (Hyperflask) 6 Number of 293T cells (Takara), a total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5 mL Antibiotic-Antimycotic (100×)), at 37 °C with 5% CO 2 Incubate for 24 hours in the incubator. 320 μg of plasmid (PTK881-EF1α-N6-MDE-8-1: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6), 320 μg of plasmid (PTK881-EF1α-C11-1: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6) DMEM basal medium was added to 960 μg PEI tube, vortexed, and equilibrated at room temperature for 10 minutes. 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. Place the multilayer cell culture flask at 37 °C with 5% CO 2 After 3 days in the incubator, the cell culture supernata...

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PUM

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Abstract

The invention discloses a construction method and application of a bispecific chimeric antigen receptor recombinant gene for treating HIV infection. The bispecific chimeric antigen receptor includes anti-CD32a single-chain antibody and anti-HIV gp120 single-chain antibody The coding nucleotide sequence of the anti-CD32a single-chain antibody and the coding nucleotide sequence of the anti-HIV gp120 single-chain antibody are connected in series in the bispecific chimeric antigen receptor coding gene. The bispecific chimeric antigen of the present invention targets the fusion protein gp120, a marker for HIV replication and proliferation, and the membrane protein CD32a, a marker for latency, to design dual-target CAR-T cells, which can completely eliminate HIV-infected target cells, It has great clinical promotion value.

Description

technical field [0001] The invention relates to the field of medicine and biology, and specifically refers to a bispecific chimeric antigen receptor for treating HIV infection, its coding gene, its construction method and its application. Background technique [0002] AIDS (AIDS) is a very harmful infectious disease caused by HIV infection. The main target of HIV attack is CD4+ T lymphocytes, destroying the cells in large quantities, leading to immune dysfunction, prone to infection, malignant tumors, and high mortality rate. Although new anti-HIV drugs and other treatment technologies have greatly improved the survival rate of AIDS patients and prolonged the survival time. However, the high emergence rate of HIV, the latent virus pool that cannot be cleared by antiviral drugs, and incomplete immune reconstitution are still three major scientific challenges. [0003] Highly Active Antiretroviral Therapy (HAART) is the first revolution in the history of HIV / AIDS treatment, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P31/18
CPCC07K14/7051C07K16/1063C07K16/283C12N15/86C12N5/0636A61K35/17A61P31/18C07K2319/02C07K2319/03C07K2319/33C07K2317/622C12N2510/00C12N2740/15043
Inventor 张同存顾潮江
Owner WUHAN BIO RAID BIOTECH CO LTD
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