Immunocompetent cells modified by dual chimeric antigen receptor genes based on CD19 and CD70 and application thereof

A technology of chimeric antigen receptors and immune cells, applied to genetically modified cells, cells modified by introducing foreign genetic material, blood/immune system cells, etc., can solve the problems of unsatisfactory long-term effects and avoid escape , Enhance long-term immune effect, strong tumor effect

Active Publication Date: 2019-06-14
BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the unsatisfactory long-term effect of single targeting in the current CAR-T technology treatment of tumors, and the fact that the tumor microenvironment affects the therapeutic effect of CAR-T technology, the present invention Provides a CD19 and CD70-based dual chimeric antigen receptor gene-modified immune cells and its application. The invent

Method used

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  • Immunocompetent cells modified by dual chimeric antigen receptor genes based on CD19 and CD70 and application thereof
  • Immunocompetent cells modified by dual chimeric antigen receptor genes based on CD19 and CD70 and application thereof
  • Immunocompetent cells modified by dual chimeric antigen receptor genes based on CD19 and CD70 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Construction of Chimeric Antigen Receptor

[0080] (1) Synthesize Secretory signal peptide, CD19 or CD70 antigen binding domain, CD8α and / or CD28 transmembrane domain, CD28 signaling domain, CD27 signaling domain, CD3ζ signaling domain, 2A sequence and Caspase 9 domains, such as figure 1 As shown, namely Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9 and Secretory-CD70 scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9; the specific sequences are as follows:

[0081] The nucleotide sequence of Secretory-CD19 scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9 is shown in SEQ ID NO.5.

[0082] The nucleotide sequence of Secretory-CD70 scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9 is shown in SEQ ID NO.7.

Embodiment 2

[0083] Example 2 lentiviral packaging

[0084] (1) Use 293T cells and culture for 17-18 hours;

[0085] (2) Add fresh DMEM containing 10% FBS;

[0086] (3) Add the following reagents to a sterile centrifuge tube: Take DMEM from each well, add helper DNA mix (pNHP, pHEF-VSV-G) and pTYF DNA vector, and vortex;

[0087] (4) Add Superfect or any transgenic material to the centrifuge tube, and let stand at room temperature for 7-10 minutes;

[0088] (5) Add the DNA-Superfect mixture in the centrifuge tube to each cultured cell drop by drop, and vortex to mix well;

[0089] (6) 37°C 3% CO 2 Cultivate in the incubator for 4-5 hours;

[0090] (7) Aspirate the culture fluid of the culture medium, wash the culture medium with 293 cell culture fluid, and add the culture fluid to continue culturing;

[0091] (8) Return the culture medium to 3% CO 2 Incubate overnight in an incubator, and observe the transfection efficiency with a fluorescence microscope the next morning.

Embodiment 3

[0092] Example 3 Purification and Concentration of Lentivirus

[0093] 1) Virus purification

[0094] Remove cell debris by centrifugation at 1000g for 5 minutes to obtain virus supernatant, filter the virus supernatant with a 0.45 micron low protein binding filter, divide the virus into small portions, and store at -80°C;

[0095] Typically, transfected cells can produce 10 6 to 10 7 Transducing Units Titrated lentiviral vectors.

[0096] 2) Concentrate the lentiviral vector with a Centricon or similar filter

[0097] (1) Add virus supernatant to Centricon or similar filter tube, then centrifuge at 2500g for 30 minutes;

[0098] (2) Shake the filter tube, then centrifuge at 400g for 2 minutes, and collect the concentrated virus into the collection cup. Finally, pool the virus in all tubes into one centrifuge tube.

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Abstract

The invention relates to an immunocompetent cells modified by dual chimeric antigen receptor genes based on CD19 and CD70 and an application thereof. The dual chimeric antigen receptors include the chimeric antigen receptor CD19 and the chimeric antigen receptor CD70. The chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signal conducting regionand a CD3 zeta signal conduct domain, and an inducible suicide fusion domain connected in series. The chimeric antigen receptor of the present invention specifically recognizes tumor surface antigensCD19 and CD70, compared to the use of other single chimeric antigen receptor T cell, the combination of two antigens is used to target CAR-T cells so that the treatment effect is better, CD19 escapeis differently generated, and the disease is easily relieved.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a CD19 and CD70-based dual chimeric antigen receptor gene-modified immune cell and its application, specifically a chimeric antigen based on tumor-specific targets CD19 and CD70 Construction method of receptor T (CAR-T) cell technology and its application in anti-tumor therapy. Background technique [0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) has become one of the most promising tumor immunotherapy. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen-binding region, an extracellular hinge region, a transmembrane region, and an intracellular signal transduction region. Usually, CAR contains the single chain fragment variable (Single chain fragment variable, scFv) region of the antibody or the binding domain specific to the tumor associated antig...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N7/01C07K16/28A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C07K2319/03C07K2317/622C07K16/2803C07K16/2875A61K2039/5156A61K39/001112C07K14/7051A61K39/001138A61K2039/505A61K2039/5158C12N2510/00C12N5/0636C12N15/86C12N2740/16043
Inventor 王佳兴
Owner BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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