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CD19 targeting chimeric antigen receptor and application thereof

A chimeric antigen receptor and application technology, applied in the field of biomedicine, can solve the problem that the immune system cannot remove tumors well, reduce immunogenicity, etc., and achieve the effects of prolonging survival time, high safety and high expression

Active Publication Date: 2020-12-15
JUVENTAS CELL THERAPY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Tumor cells produce immune escape by down-regulating the expression of molecules involved in T cell recognition and antigen response, reducing immunogenicity, etc., so that the body's immune system cannot clear the tumor well

Method used

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  • CD19 targeting chimeric antigen receptor and application thereof
  • CD19 targeting chimeric antigen receptor and application thereof
  • CD19 targeting chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Construction of lentiviral vector

[0108] Artificially synthesized fragments containing the CAR structure described in this application (the amino acid sequence and nucleotide sequence are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively), and constructed into a lentiviral vector (manufacturer: SBI, article number: CD500-CD800), the obtained expression vector was named CNCT19 expression vector, and then transfected to obtain lentivirus according to the method described in its instruction manual, and the virus transfection titer was determined by flow cytometry to confirm that a functional lentiviral vector was obtained .

Embodiment 2

[0109] Example 2. Preparation of lentivirus-infected T cells

[0110] Infection experiments were performed according to conventional methods known to those skilled in the art. A brief description of the infection steps is as follows:

[0111] 1. Sorting T Cells

[0112] Peripheral blood mononuclear cells (PBMC) were isolated from blood samples collected from subjects, and T cells were then sorted from PBMC cells.

[0113] 2. Activate T cells

[0114] Separated T cells were treated with complete lymphocyte culture medium (Xvivo15 medium + 5% FBS + 100IU / ml IL-2 or Xvivo15 medium + 5% FBS + 20ng / ml IL-21 + 10ng / ml IL-7) Resuspend to make the final concentration (1~2)×10 6 cells / ml, and add 5-10 μl of CD3 / CD28 stimulating magnetic beads, mix well and place in the incubator for culture, the culture condition is 37℃+5%CO 2 , the incubation time is at least 24 hours.

[0115] 3. Lentivirus Infection of T Cells

[0116] Take out the activated cultured T cells, add polybrene (p...

Embodiment 3

[0119] Example 3. Detection of the expression of CAR molecules on the surface of CNCT19 cells

[0120] The experimental steps are as follows:

[0121] (1) Centrifuge the CNCT19 cell suspension at 300g for 5 minutes, discard the supernatant, add sheath fluid and resuspend until the viable cell density is 0.5~1×10 7 cells / mL.

[0122] (2) Take 2 flow tubes for each sample, labeled as 1~2 tubes, the first tube is a blank control, no need to add antibodies, add 10 times diluted Alexa Fluor to the second tube ® 647-Goat anti-mouse IgG F(ab') 2 Antibody (manufacturer: Jackson, product number: 115-605-072) 10 μL.

[0123] (3) Add 100 μl of cell suspension to each tube, and react for 15-20 minutes at room temperature in the dark.

[0124] (4) Add 2ml of sheath fluid to each tube for washing, and centrifuge at 300g for 5 minutes.

[0125] (5) Discard the supernatant, add 20 μl of FITC-CD3 (manufacturer: Tongsheng Times, article number: Z6410047-100T) to the second tube, and incub...

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Abstract

The invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises an amino acid sequence shown in SEQ ID NO. 1. The application also provides nucleic acids encoding the chimeric antigen receptor, vectors comprising the nucleic acids, immune effector cells comprising the chimeric antigen receptor, the nucleic acid molecules and / or the vectors, methods of preparing the immune effector cells, compositions comprising the immune effector cells, and uses of the chimeric antigen receptor.

Description

technical field [0001] This application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting CD19 and its use. Background technique [0002] The clinical treatment of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and B-cell lymphoma is currently based on chemotherapy, stem cell transplantation and biological therapy. The key point that is difficult to overcome, as a new treatment strategy, cellular immunotherapy of tumors has become a research hotspot today. CD19 is expressed on the surface of almost all B cell tumor cells, while other parenchymal cells and hematopoietic stem cells are almost not expressed. [0003] Tumor cells generate immune escape by down-regulating the expression of molecules involved in T cell recognition and antigen response, reducing immunogenicity, etc., so that the body's immune system cannot clear the tumor well. Studies have found that chimeric antigen receptor T cells (CAR-T cells)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/12C12N15/85C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N5/0636C12N5/0646A61P35/00A61P35/02C12N2510/00C12N15/86C12N2740/15043C07K2319/33A61K39/001112C07K2319/03C07K2317/622C12N2740/16043C07K14/70596C07K14/70517C07K14/70521C07K14/70578A61K2039/572A61K2039/804A61K2239/48A61K39/4631A61K39/4611A61K2239/31A61K39/464412A61K2239/38A61K39/4644A61K39/4613A61K2121/00A61K2300/00C07K16/2887
Inventor 王建祥王敏吕璐璐石琳安娜王瑞刘芸
Owner JUVENTAS CELL THERAPY LTD
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