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Chimeric antigen receptor, and application thereof

A chimeric antigen receptor and antigen technology, applied in the field of tumor cell immunotherapy, can solve the problems of reduced expression of CD19, poor curative effect, easy mutation expression of CD19, etc., and achieve the effect of enhanced immune effect and high expression

Active Publication Date: 2017-11-10
SHENZHEN GENO IMMUNE MEDICAL INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chimeric antigen receptor gene-modified T cells targeting CD19 molecules (CD19CAR-T) have achieved great success in the treatment of multiple, refractory acute B-lymphoblastic leukemia, while in refractory, relapsed chronic B-lymphoblastic leukemia Significantly poorer outcomes in treatment of leukemia and B-lineage lymphoma
[0005] CN 104788573 A discloses a chimeric antigen receptor hCD19scFv-CD8α-CD28-CD3ζ and its use. The chimeric antigen receptor consists of anti-human CD19 monoclonal antibody HI19a light chain and heavy chain variable region (hCD19scFv), human The hinge region of CD8α, the transmembrane region and intracellular region of human CD28, and the intracellular region of human CD3ζ are constructed in tandem. After the CD19 in this patent is reinfused with CAR-T cells, the expression level of CD19 will decrease and it is easy to escape immune mechanism
[0006] Therefore, it is particularly important to prepare a chimeric antigen receptor that can solve the problems of easy mutation and reduced expression of CD19

Method used

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  • Chimeric antigen receptor, and application thereof
  • Chimeric antigen receptor, and application thereof
  • Chimeric antigen receptor, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Construction of Chimeric Antigen Receptor

[0055] (1) Synthesize Secretory signal peptide, CD20 antigen-binding domain, CD8α and / or CD28 transmembrane domain, CD28 signaling domain, CD127 signaling domain, IL-15Ra signaling domain and CD137 signaling through whole gene synthesis domain, CD3ζ signaling domain, 2A sequence and caspase 9 domain, such as figure 1 shown, namely

[0056] Secretory-CD20-CD28-CD127-IL-15Ra-CD137-CD3ζ-2A-FBKP.Casp9;

[0057] The nucleotide sequence SEQ ID NO.3 of the chimeric antigen receptor is as follows:

[0058]ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATCCCCGGCGACATCGTGATGACCCAGACCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGAAGCAGCAAGAGCCTGCTGCACAGCAACGGCATCACCTACCTGTACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCAGATGAGCAACCTGGTGAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAGCTGCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGGGCAGCACCAGCGGCAGCG...

Embodiment 2

[0059] Example 2: Lentiviral Packaging

[0060] (1) Culture 293T cells in a six-well plate, 1×10 6 cells / well, cultivated for 17-18 hours;

[0061] (2) Add 600 μL / well of fresh DMEM containing 10% FBS;

[0062] (3) Add the following reagents to a sterile centrifuge tube: Take 75 μL of DMEM supernatant per well, 2.7 μg of helperDNA mix (1.8 μg pNHP, 0.5 μg pHEF-VSV-G, 0.2 μg pHEFeGFP) and 0.8 μg of pTY DNA vector, vortex;

[0063] (4) Take 7 μL of Superfect from the center of each well plate, add it to the centrifuge tube and blow it 5 times, and let it stand at room temperature for 7-10 minutes;

[0064] (5) Add the DNA-Superfect mixture in the centrifuge tube dropwise to each culture well, and vortex to mix well;

[0065] (6) 37°C 3% CO 2 Cultivate in the incubator for 4-5 hours;

[0066] (7) Aspirate the culture medium of the medium, wash the medium with 1.5mL AIM-V, and add 1.5mL of AIM-V to continue culturing;

[0067] (8) Return the culture medium to 3% CO 2 Incub...

Embodiment 3

[0068] Example 3: Purification and concentration of lentivirus

[0069] 1) Virus purification

[0070] Remove cell debris by centrifugation (1000g, 5 minutes) to obtain virus supernatant, filter the virus supernatant with a 0.45 micron low protein binding filter, virus is aliquoted and stored at -80°C;

[0071] Typically, transfected cells can produce 10 6 to 10 7 Transducing Units Titrated lentiviral vectors.

[0072] 2) Concentrate lentiviral vectors with Centricon filters

[0073] (1) In a biological safety cabinet, take a Centricon tube, disinfect it once with 70% alcohol, and then wash it three times with sterile PBS;

[0074] (2) Add 18ml of virus supernatant to each Centricon P-20 filter tube, then centrifuge at 2500g for 30 minutes or until the virus volume is reduced to 0.5ml;

[0075] (3) Shake the filter tube, then centrifuge at 400g for 2 minutes to collect the concentrated virus in the collection cup. Finally, pool the virus in all tubes into one centrifuge ...

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Abstract

The invention relates to a chimeric antigen receptor, and application thereof, specifically to a construction method for a cell technology of a chimeric antigen receptor T (CAR-T) based on a tumor-specific target CD20 and application of the CAR-T to anti-cancer treatment. The chimeric antigen receptor is formed by an antigen binding domain, a transmembrane domain, a costimulatory-signal transduction domain and a CD3 zeta signal transduction structural domain through series connection, wherein the antigen binding domain binds to a tumor surface antigen, and the tumor surface antigen is CD20. The chimeric antigen receptor carries out specific gene modification on a single-chain antibody targeting to the tumor surface antigen CD20, so the modified antibody allows antigen-antibody binding force to be stronger and mutation is hard to occur; and compared with other chimeric antigen receptors and other tumor antigens, the chimeric antigen receptor provided by the invention has better effect, a high target expression level and improves the immunization effect and treatment effect of CAR-T cells.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a chimeric antigen receptor and its application, specifically a method for constructing chimeric antigen receptor T (CAR-T) cell technology based on tumor-specific target CD20 and its application in antitumor 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 antigen (tumor associated antigen, TAA), which communicates with t...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N7/01C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K14/70578C07K14/70596C07K16/2887C07K2317/622C07K2319/00C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N7/00C12N9/6472C12N2510/00C12N2740/15021C12Y304/22062
Inventor 李昱琛张政维
Owner SHENZHEN GENO IMMUNE MEDICAL INST
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