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RVG-CD70 CAR-T cells and preparation method and application thereof

An RVG-CD70CAR-T, cell technology, applied in the fields of molecular biology and cell biology, can solve the problem of difficult surgical large-scale complete resection, and achieve the effect of increasing the specificity of T cell killing and high-efficiency tumor killing activity

Active Publication Date: 2020-05-12
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Brain tumors include primary brain tumors and brain metastases. The best treatment method is complete surgical resection. However, due to the unresectable nature of normal brain tissue and the characteristics of extensive invasive growth of malignant tumors into the brain, large-scale complete surgical resection is recommended. extremely difficult

Method used

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  • RVG-CD70 CAR-T cells and preparation method and application thereof
  • RVG-CD70 CAR-T cells and preparation method and application thereof
  • RVG-CD70 CAR-T cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Preparation of lentiviral expression vector

[0052] Gene synthesis RVG-SCFV(anti CD70)-CD8 TM - 4-1BB-CD3ζ fusion gene sequence, the gene sequence is shown in SEQID NO.8, which is connected to the PLV vector through enzyme digestion transformation, and the upstream of the gene is the EF-1α promoter. Transform the DH5α Escherichia coli strain with the vector, screen with ampicillin, obtain positive clones, extract the plasmid, identify the clones by enzyme digestion, and obtain PLV-RVG-SCFV(anti CD70)-CD8 TM -4-1BB-CD3ζ lentiviral packaging vector, see figure 1 .

Embodiment 2

[0053] Example 2: Preparation of lentivirus

[0054] 24 hours before transfection, use about 8×10 per bottle 6 293T cells were inoculated into T75 culture flasks. Make sure that the cells are about 80% confluent and evenly distributed in the culture flask for lentiviral packaging.

[0055] Prepare Plasmid and Transfection Reagent Dilutions

[0056] 1. Vortex to mix the PEI transfection reagent.

[0057] 2. Prepare 2 centrifuge tubes, and prepare plasmid and transfection reagent dilutions in the following order.

[0058]

[0059]

[0060] 3. Mix well.

[0061] 4. Add the transfection reagent dilution (centrifuge tube 2) into the plasmid DNA solution (centrifuge tube 1), and mix thoroughly immediately. Note that the join order is very important.

[0062] 5. Incubate the transfection mixture at room temperature for 15-20 minutes.

[0063] 6. Add 1ml of the transfection mixture into the well-prepared 293T cell culture flask, and gently blow and aspirate the medium to ...

Embodiment 3

[0067] Example 3: Preparation of RVG-CD70 CAR-T cells

[0068] Take 0.5ml of blood for rapid detection of pathogenic microorganisms to rule out microbial infections such as HBV, HCV, HDV, HEV, HIV-1 / 2, Treponema pallidum and parasites. Peripheral blood mononuclear cells (PBMC) were collected from the patients by an apheresis machine. Configure complete growth medium, add 5% autologous AB or FBS to PBS, IL-2 concentration is 20ng / ml, and dilute the isolated PBMC with medium to 2×10 6 / ml, take 50u1 flow cytometry to detect the purity of T cells in PBMC. On day 0, configure buffer1, add 1% FBS to PBS, shake the beads for 30s or manually shake them up and down for 5 minutes, take out the CD3 / CD8 beads according to the ratio of beads to T cells 3 to 1, put them in a 1.5ml EP tube, add 1ml Wash the beads with Buffer1, then use a magnet to suck the beads from the EP tube for 1 min, discard the washing solution, repeat twice, then use the medium to resuspend the beads to the origin...

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Abstract

The invention belongs to the field of molecular biology and cell biology, and particularly relates to CAR-T cells which can penetrate through blood-brain barriers and can specifically recognize CD70,and a preparation method. RVG-SCFV (Anti CD70)-CD8<TM>-4-1BB-CD3 zeta molecules are expressed in T cells. A CD70 antibody is used for constructing the CAR-T cells, viewpoint that CD70 molecules are used as a target antigen is put forward, when the constructed CAR-T cells are in contact with tumor cells expressing the CD70 molecules, the distance can be drawn to be the smallest, and the killing specificity of the T cells can be increased; and the prepared RVG-CD70 CAR-T cells have RVG protein which assists in penetrating through the blood-brain barrier, can be efficiently positioned at the brain part to exert tumor killing activity, and can be used for preparing CAR-T cell medicines for treating cerebral tumors high in CD70 molecule expression.

Description

technical field [0001] The present invention belongs to the fields of molecular biology and cell biology, and specifically relates to a CAR-T cell targeting the brain with RVG protein and specifically recognizing CD70 and a preparation method thereof, and its use in treating brain tumors related to highly expressed CD70 molecules application in medicine. Background technique [0002] The hypofunction of the immune system of cancer patients due to radiotherapy and chemotherapy is an important reason for the recurrence of the disease, and it is also a major problem that plagues the medical field. Chimeric antigen receptor T-cell immunotherapy (CAR-T), with its unique anti-tumor advantages, has become a hot spot in global biotechnology competition. CAR-T cells combine the single-chain variable region (SCFV) of an antibody that can recognize a certain tumor antigen with CD8 TM , 4-1BB, CD3-ζ chain and other intracellular regions are coupled into a chimeric protein, which is tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C12N15/867A61K39/00A61P35/00
CPCC12N5/0636C07K16/2875C07K14/7051C07K14/70517C07K14/70578C12N15/86A61K39/001102A61P35/00C12N2510/00C07K2317/622C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107A61K2039/5156A61K2039/80
Inventor 纪锋郭志刚孙裕铃张凡景丽李恩洁马颖胡志刚
Owner NANJING NORMAL UNIVERSITY
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