Chimeric antigen receptor T cells as well as preparation method and application thereof

A chimeric antigen receptor and cell technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of strict conditions for patient enrollment and patients who cannot be enrolled in group therapy.

Inactive Publication Date: 2017-05-31
SHENZHEN PREGENE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although very good results have been achieved in clinical treatment, especially for the treatment of leukemia, the enrollment conditions for these patients are relatively strict, and patients with v...

Method used

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  • Chimeric antigen receptor T cells as well as preparation method and application thereof
  • Chimeric antigen receptor T cells as well as preparation method and application thereof
  • Chimeric antigen receptor T cells as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Pre-Lenti-EF1-CD19CAR vector construction

[0029] 1. Synthetic ScFV gene: the heavy chain amino acid codon encoding CD8 signal peptide, sequence number CAA74659.1 was obtained by PCR method and optimized to nucleotide, (G4S) 3 The hinge region and the amino acid codon of the light chain with the sequence number CAA74660.1 are optimized as a nucleotide sequence of a nucleotide fragment of the ScFV gene of the CD19 single-chain antibody, the base sequence of which is shown in SEQ ID NO:1.

[0030] 2. Synthesize the third-generation CAR structural gene, and obtain the nucleosides of the CAR structural gene encoding the nucleotide fragments of the CD8 hinge region, CD28 transmembrane region, cytoplasmic region, 4-1BB cytoplasmic region and CD3ζ cytoplasmic region by PCR Acid fragment, its base sequence is shown in SEQID NO:2.

[0031] 3. The synthetic ScFV gene and the third-generation CAR structural gene were spliced ​​by overlapping PCR to obtain the CD19CAR ...

Embodiment 2

[0066] Embodiment 2: cell killing experiment

[0067] 1. Lentiviral packaging

[0068] (1) 293T cells were cultured at 37°C, 5% CO 2 In the incubator, the medium is DMEM / 10% FBS.

[0069] (2) One day before packaging the virus, trypsinize 293T cells, 1×10 7 Cells / well were seeded in 10 cm Petri dishes.

[0070] (3) When transfecting cells, in addition to the Pre-Lenti-EF1-MCS-CD19CAR plasmid, each plasmid should be co-transfected with packaging plasmids psPAX2 and pMD2.0G. Among them, Pre-Lenti-EF1-MCS-CD19CAR uses 5 μg, psPAX2 uses 3.75 μg, and pMD2.0G uses 1.25 μg. For transfection, add the mixture of the above three plasmids to 500 μl opti-MEM medium, add 25 μl Lipofectamine 2000 reagent to 500 μl opti-MEM medium in another microcentrifuge tube, and then add the diluted transfection reagent dropwise On top of the diluted plasmid, mix well, centrifuge, and stand at room temperature for 20 minutes. Finally, add the mixture of plasmid and transfection reagent to a 10cm cu...

Embodiment 3

[0108] Embodiment 3: animal experiments

[0109] 1. Mouse strain: it is NSG immunodeficiency mouse strain (lack of T cell, B cell and NK cell function). 2. Tumor inoculation: using Raji lymphoma cell line, 1x10 6 Cells / 200ul PBS tail vein injection (6 mice).

[0110] 3. CART injection: 2 days after Raji tumor cell inoculation, Control or CD19CART cells (1x10 7 cells / 200ul PBS, a group of 3 mice), see Figure 6 . Depend on Figure 6 It can be seen that the control CART cannot effectively inhibit the growth of lymphoma cells in the liver (the white plaques are tumors formed by lymphoma cells), while CD19 CART can effectively inhibit lymphoma.

[0111] 4. Observe every 2 days, measure the body weight of the mice, until 2 weeks after the CART cell injection, the mice in the control group (Control) begin to appear hindlimb paralysis, and die one after another. Dissect and observe, record the time of death, and sort out the survival curve, see Figure 7 . Depend on Figure 7...

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Abstract

The invention discloses chimeric antigen receptor T cells as well as a preparation method and an application thereof. The surfaces of the T cells express chimeric antigen receptor CD19 CAR genes, with the base sequence shown as SEQ ID NO: 3. The invention further discloses a preparation method and an application of the chimeric antigen receptor T cells. Two kinds of co-stimulatory signals are added into the structures of third-generation CD19 CAR vectors to construct lentiviral vectors, the effects of the third-generation CD19 CART are observed in cell and animal experiments, and the third-generation CD19 CART is proved to have the capability of effectively killing tumor cells.

Description

technical field [0001] The invention relates to a chimeric antigen receptor T cell and its preparation method and application. Background technique [0002] Chimeric Antigen Receptor T cells (CART) are genetically modified to enable the single-chain variable region of a monoclonal antibody that specifically recognizes the target antigen [0003] (scFv) is expressed on the surface of T cells, and the scFv is coupled to the activation and proliferation signal domain in the artificially designed T cells through the transmembrane region. In this way, the specific recognition of the target antigen by the monoclonal antibody is combined with the function of the T cell to produce a specific killing effect, and CART can kill the target cell in a non-MHC-restricted manner. Now CAR-T lymphocyte technology has been developed to the third generation. In 1989, the scientist Zelig Eshhar proposed the concept of CART. In the subsequent development of CART technology, it has mainly underg...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00A61P35/02
CPCC12N5/0636A61K35/17C12N15/86C12N2510/00C12N2740/15043
Inventor 栗红建何昱余祥姜冬冬赵杨杨孟邑芳
Owner SHENZHEN PREGENE BIOPHARMA CO LTD
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