A bispecific chimeric antigen receptor, gene, construction method and application thereof for the treatment of hematological tumors complicated with HIV infection

A chimeric antigen receptor and bispecific technology, applied in the field of medicine and biology, can solve the problems of large side effects, high mortality of patients, and easy recurrence, and achieve the effect of accelerated clearance

Active Publication Date: 2021-02-09
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main treatment for lymphoma is chemotherapy, which has large side effects and is prone to relapse, and there is no available therapy
However, patients with HIV combined with malignant tumors are generally in serious condition, their immune system is severely damaged, their disease progresses rapidly, and their mortality rate is high. For this, how to choose an effective treatment plan is imminent

Method used

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  • A bispecific chimeric antigen receptor, gene, construction method and application thereof for the treatment of hematological tumors complicated with HIV infection
  • A bispecific chimeric antigen receptor, gene, construction method and application thereof for the treatment of hematological tumors complicated with HIV infection
  • A bispecific chimeric antigen receptor, gene, construction method and application thereof for the treatment of hematological tumors complicated with HIV infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of PTK881-EF1α-C5 and PTK881-EF1α-C6 plasmids

[0043] 1. The sequences SP1-N6 (nucleotide sequence SEQ ID NO: 1), SP2-FMC63-28Z (nucleotide sequence SEQ ID NO: 2) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the synthesized sequences were cloned into pUC57 on the carrier.

[0044] 2. Using the human cDNA library as a template, design primers to amplify the fragments CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, CD8 transmembrane region, CD3ζ intracellular signaling domain, and Primer complementation method to obtain Strep II, connecting peptide 3×G 4 S, P2A;

[0045] 3. Using Overlap PCR technology to combine SP1-N6, Strep II, and connecting peptide 3×G 4 S, SP2-FMC63-28Z and CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, and CD3ζ intracellular signaling domain were sequentially amplified and connected to obtain the code of chimeric antigen recepto...

Embodiment 2

[0052] Example 2: Preparation and sequencing of plasmids

[0053] 1. Preparation of plasmid

[0054] Inoculate the DH5α strains containing plasmids PTK881-EF1α-C5-1 and PTK881-EF1α-C6-1 into 250 mL of LB culture medium containing 100 μg / mL kanamycin, and culture overnight at 37°C and 220 rpm. The culture solution was centrifuged at 6000g for 20min at 4°C, and the supernatant was discarded.

[0055] 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 .

[0056] 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.

[0057] Add 120mL Buffers P3 to the centrifuge bottle, put the cap on the roller mixer, slowly increase the speed to the maximum speed of...

Embodiment 3

[0070] Example 3, Preparation of Lenti3-C5, Lenti3-C6 Lentiviral Vectors and Live Droplet Detection

[0071] 1. Preparation of lentiviral vector

[0072] 130.0~140.0×10 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. Mix 320 μg of plasmids (PTK881-EF1α-C5: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6 and PTK881-EF1α-C6: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5 : 6) The DMEM basal medium was added to two 960μg PEI tubes, vortexed, and equilibrated at room temperature for 10 minutes. Mix the above-mentioned two tubes of 35mL PEI and plasmid mixture with 525mL DMEM complete medium respectively, and replace them 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 supernatant was collected.

[0073] After th...

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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 complicated with hematological tumors. The chimeric antigen receptor consists of signal peptide, HIV gp120 antigen-specific single chain antibody and The anti-CD19 single-chain antibody is connected in sequence, and then formed in series with the CD28 transmembrane region, CD28 intracellular domain (ICD), 4-1BB co-stimulatory domain, and CD3ζ intracellular signaling domain, or the chimeric antigen is stimulated The body consists of signal peptide, HIV gp120 antigen-specific single chain antibody and CD28 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, CD3ζ intracellular signaling domain (the first CAR) and signal peptide, anti-CD19 monoclonal Chain antibody, CD8 transmembrane region, CD28-ICD, 4-1BB co-stimulatory domain, CD3ζ intracellular signaling domain (second CAR), first CAR and second CAR are sequentially connected in parallel.

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 blood tumors complicated with HIV infection, its coding gene, its construction method and its application. Background technique [0002] After the human body is infected with HIV, CD4+ T lymphocytes are attacked by the virus, resulting in reduced immunity of the body, prone to various viral infections or malignant tumor diseases, and lymphoma is one of them. The risk of AIDS-related lymphoma is 165 times that of the general population. Malignant lymphoma is currently one of the leading causes of death in AIDS patients. [0003] According to the opinions of oncologists, patients with HIV complicated with malignant tumors were treated with a medical treatment plan, and the treatment plan was anti-tumor combined with ART treatment; According to the results of drug susceptibility test, etc., give patients immune suppor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K39/42A61P35/00A61P31/18
CPCA61K39/42A61K2039/505A61K2039/5156A61P31/18A61P35/00A61K39/001112A61K2039/804C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K16/1063C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 顾潮江张同存
Owner WUHAN UNIV OF SCI & TECH
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