CD19 specific chimeric antigen receptor and encoding gene and application thereof

A chimeric antigen receptor and specific technology, applied in the field of cellular immunity, can solve the problems of poor tolerance, strong toxic and side effects, and high recurrence rate

Inactive Publication Date: 2016-11-16
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Large doses of radiotherapy and chemotherapy have strong toxic and side effects, poor tolerance of patients, and greatly reduced immunity. Although it can be relieved, the recurrence rate is high
Moreover, the source of hematopoietic stem cells is scarce, and the success rate of matching is low

Method used

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  • CD19 specific chimeric antigen receptor and encoding gene and application thereof
  • CD19 specific chimeric antigen receptor and encoding gene and application thereof
  • CD19 specific chimeric antigen receptor and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of CD19-specific chimeric antigen receptor retroviral vector

[0032] A CD19-specific chimeric antigen receptor proposed in the present invention is composed of a human anti-CD19 single-chain antibody, the CH2CH3 of human antibody IgG4, the transmembrane region and the intracellular signal structure of CD28, the intracellular signal structure of CD137, and the CD3ζ. The intracellular signal structure of the tandem is composed of tandem, and its amino acid sequence is shown in SEQ ID NO.1.

[0033] 1. Using the FMC62 mouse anti-human CD19 monoclonal antibody sequence published in 1991, select V H and V L The sequence of scFv is used to design the scFv sequence in the CAR vector. The codons are further optimized by GenScript Biotechnology's OptimumGeneTM gene design software, and NcoI and BAMHI restriction sites are added to both ends of the optimized sequence. After optimization The sequence of the gene was synthesized by Nanjing GenScript Biote...

Embodiment 2

[0037] Example 2: Preparation of CD19-specific chimeric antigen receptor-modified T lymphocytes

[0038] 1. Packaging of retrovirus containing CD19-specific chimeric antigen receptor

[0039] The retroviral packaging vector pRD114 and CD19-scFv-CH2CH3-CD28-CD137-CD3ζ retroviral vector were co-transfected into 293T cells using the instructions in the endotoxin-free plasmid extraction kit (Tiangen Biotechnology). The cell supernatant was collected 48h after transfection, centrifuged at 4000rpm for 10min, and the cells and cell debris in the supernatant were removed. Filter through a 0.45 μm filter, aliquot and store for later use.

[0040] 2. Preparation of T lymphocytes

[0041] 20 mL of fresh anticoagulant from healthy volunteers was taken, and peripheral blood mononuclear cells (PBMC) were isolated with lymphatic separation medium (GE). The isolated cells were stimulated with CD3 and CD28 plates for 48 hours, and induced with T lymphocyte medium GT-T551 (TAKARA) plus 3% au...

Embodiment 3

[0046] Example 3: Killing effect of chimeric antigen receptor CD19-specific CAR-T cells on CD19-related tumors

[0047] 1. Detection of lethality of CAR-T cells to tumors

[0048] Adjust the lymphoma cells with medium to 5 × 10 6 / mL, 50 μL per well, according to the ratio of effector cells to target cells 1:4, add 2.5×10 CD19 positive lymphoma cells 5 After the cells are completely attached, collect T cells and CAR-T cells, and adjust the cell concentration to 1.25×10 6 / mL, 50 μL per well, and then flow cytometry (cultured for 24h) and MTT detection (cultured for 12h) were performed.

[0049] First, cells were collected for CD3 and CD19 staining, and flow cytometric analysis of tumor cells (CD19 + ) and T cells (CD3 + ), the result is as Figure 5 shown. Depend on Figure 5 It can be seen that the chimeric antigen receptor CD19-specific CAR-T cells obtained in the present invention can effectively kill lymphoma cells.

[0050] Next, in the same experiment, discard th...

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PUM

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Abstract

The invention discloses a CD19 specific chimeric antigen receptor. The CD19 specific chimeric antigen receptor is formed by connecting a single-chain antibody of human antibody CD19, CH2CH3 of a human antibody IgG4, a transmembrane domain and intracellular signal structure of CD28, an intracellular signal structure of CD137 and an intracellular signal structure of CD3zeta in series. The invention further discloses an amino acid sequence and a nucleotide sequence of the chimeric antigen receptor. The invention further discloses application of the chimeric antigen receptor in preparation of medicine for treating CD19 related tumors. According to the application, T-lymphocyte is modified, and the modified lymphocyte can be used for treating the CD19 related tumors.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, in particular to a CD19-specific chimeric antigen receptor and its encoding gene and application. Background technique [0002] B lymphocyte tumors are malignant hematological diseases originating from B cells, mainly including acute lymphoid leukemia (ALL), non-Hodgkin lymphoma (NHL) and most chronic lymphoid leukemia (CLL) derived from B cells. . Among them, the incidence of ALL is the highest, and there is a trend of increasing year by year. Current treatment is mainly through systemic chemotherapy and transplantation of hematopoietic stem cells. High-dose radiotherapy and chemotherapy have strong toxic and side effects, poor patient tolerance, and greatly reduced immunity. Although it can be alleviated, the recurrence rate is high. Moreover, the source of hematopoietic stem cells is scarce, and the matching success rate is low. [0003] Chimeric antigen receptor modified T cell ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K35/17A61P35/00
CPCC07K16/2803A61K35/17C07K16/18C07K2317/622C07K2319/02C07K2319/33
Inventor 宋晓彤许国贞刘振云
Owner SINOBIOWAY CELL THERAPY CO LTD
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