Chimeric antigen receptor (CAR) against CD20 antigen and application thereof

A technology of chimeric antigen receptors and antigens, applied in the field of genetic engineering, can solve problems such as large demand for drugs, drug resistance in patients, and short half-life

Active Publication Date: 2019-02-05
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although rituximab can clear CD20-expressing B lymphocytes to a certain extent, its half-life in the body is short, the drug demand is large, and many patients will eventually develop drug resistance
Whether it is possible to use the specificity of rituximab to target CD20-positive blood diseases, and to modify the polypeptide structure of rituximab with a chimeric antigen receptor (CAR) to obtain safe and effective CAR-T cells targeting CD20. There is no research report, and whether rituximab is the best combined CAR peptide for CD20 monoclonal antibody combination recognition also needs further verification

Method used

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  • Chimeric antigen receptor (CAR) against CD20 antigen and application thereof
  • Chimeric antigen receptor (CAR) against CD20 antigen and application thereof
  • Chimeric antigen receptor (CAR) against CD20 antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of chimeric antigen receptor virus containing CD20scFv

[0059] In order to construct a more effective chimeric antigen receptor against CD20, we used three murine antibodies to construct its scFV and designed CD20R, CD20A, CD20U and CD20R3-8h, CD20R2-8h, CD20R3-G4h, CD20R2-G4h seven groups of CAR structures ,like figure 1 shown.

[0060] 1 Synthesis of gene sequences of CD20-targeting chimeric antigen receptors containing different scFVs

[0061] Synthesize different single-chain antibody ScFv containing anti-human CD20 antigen in turn, IgG4 or human CD8 hinge region (also known as hinge region), CD28 transmembrane region (abbreviated as TM), CD28 and / or CD137 and CD3 co-stimulatory signal combination The resulting chimeric antigen receptor has a structure such as figure 1 shown.

[0062] 2 Construction of lentiviral vectors expressing chimeric antigen receptors

[0063] The following primers were designed and synthesized by Nanjing GenScrip...

Embodiment 2

[0077] Example 2 Expression of CD20 in different tumor cells

[0078] Tumor cells Raji-Luc, Ramos-Luc, and K562-Luc cultured for 10 days were collected into 10ml centrifuge tubes, centrifuged at 300g / min for 5min, and the supernatant was discarded to collect the cells; Resuspend the cells in PBS solution, divide the collected cells into 100 μl tubes, add 5 μl PE-CD20 antibody (BD company) to each tube of cells, incubate at 4°C for 30 min, wash twice with PBS solution, and perform the test on a flow cytometer. detection.

[0079] The result is as figure 2 As shown, Raji-Luc and Ramos-Luc are positive cells with high expression of CD20, and K562-Luc is a negative cell of CD20 and does not express CD20.

Embodiment 3

[0080] Example 3 Detection of CAR Transfection T Cell Ability and CAR-T Cell Killing Ability

[0081] 1. Detection of the ability of CD20-CAR to transfect T lymphocytes

[0082] 1 Isolation of Human Peripheral Blood Mononuclear Cells

[0083] Collect about 60ml of peripheral blood with anticoagulant-added blood collection tubes, divide into 50ml centrifuge tubes, add 7.5ml of hydroxyethyl starch to dilute; naturally settle at room temperature for about 30min, collect the upper plasma and centrifuge; then resuspend the precipitate with normal saline, Add it to the lymphocyte separation medium at a volume ratio of 1:1, and perform gradient centrifugation; after centrifugation, take the second white lymphocyte layer and wash it twice with normal saline to obtain PBMC cells, add RPMI 1640 containing 10% FBS to complete culture medium.

[0084] 2 lentiviral vector infection of T lymphocytes

[0085] Mononuclear cell PBMCs obtained in step 1 were activated by adding anti-CD3 mono...

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Abstract

The invention belongs to the field of genetic engineering, in particular to a chimeric antigen receptor (CAR) against a CD20 antigen and an application thereof. The CAR against the CD20 antigen is characterized in that the CAR is formed by polypeptides, namely single-chain variable fragment (scFv) for identifying the CD20 antigen, a hinge region, a transmembrane region and intracellular signals ina sequentially connected mode; and the amino acid sequence of the polypeptides (scFv) for identifying the CD20 antigen is shown as SEQ ID NO.14. After the CAR against the CD20 antigen provided by theinvention is expressed in immune cells, tumor target cells expressing the CD20 antigen can be effectively removed, it can be maintained that targeted CD20 molecules are combined with CD20 molecules on the cell surface to achieve the effect of killing B lymphocyte, the ability of multiplication and the ability of CAR-T to kill tumors can be enhanced, the toxic and side effects on antigen-negativecells are avoided, and the CAR can be used for targeted treatment of the tumors.

Description

technical field [0001] The invention belongs to the field of genetic engineering, relates to a chimeric antigen receptor for anti-CD20 antigen and its application, and also relates to a lentiviral vector containing the chimeric antigen receptor for anti-CD20 antigen and its application. Background technique [0002] Chronic lymphocytic leukemia (CLL) is a clonal proliferative neoplasm of mature B lymphocytes, characterized by the accumulation of lymphocytes in peripheral blood, bone marrow, spleen and lymph nodes. Many high-risk patients are prone to relapse during treatment, and there are certain difficulties in treatment. [0003] Non-Hodgkin's lymphoma (NHL) is a general term for a group of independent diseases with strong heterogeneity, and it ranks within the top 10 of common malignant tumors. At present, the incidence rate shows an obvious upward trend in different age groups. [0004] CD20 antigen is a B cell differentiation antigen, which is only located in pre-B c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N15/66C12N5/10A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K16/2887C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N15/66C12N15/86C12N2740/15043
Inventor 不公告发明人
Owner CHONGQING PRECISION BIOTECH CO LTD
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