Chimeric antigen receptor immune cell provided with safety switch as well as preparation method and application of chimeric antigen receptor immune cell

A chimeric antigen receptor and safety switch technology, applied in the field of biomedicine, can solve problems such as cell mutation and canceration

Inactive Publication Date: 2017-05-31
AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although CAR-T technology has been proven to be highly effective by most domestic and foreign preliminary clinical studies, it is similar to the conventional lymphoma / leukemia radiotherapy and chemotherapy treatment process, and the side effects are still not to be underestimated; Similar to the treatment, the application of CAR-T cells needs to be combined with cytokines and other related treatments, such as chills, fever, leukopenia, tumor lysis syndrome, cytokine storm, hypoimmunoglobulinemia caused by B cell deficiency, etc. It is a common adverse reaction; in addition, the integration of foreign genes also has the risk of causing cell mutation or even cancer

Method used

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  • Chimeric antigen receptor immune cell provided with safety switch as well as preparation method and application of chimeric antigen receptor immune cell
  • Chimeric antigen receptor immune cell provided with safety switch as well as preparation method and application of chimeric antigen receptor immune cell
  • Chimeric antigen receptor immune cell provided with safety switch as well as preparation method and application of chimeric antigen receptor immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Synthesis of CAR expression cassette and construction of expression vector

[0052]According to the amino acid sequence and coding sequence of each component of CAR, splicing into the entire fused amino acid sequence and coding DNA expression cassette, the full-length sequence contains anti-hCD20scFv (SEQ ID NO: 2 or SEQ ID NO: 6) and human CD28 Transmembrane and intracellular segment (SEQ ID NO:3 or SEQ ID NO:7), CD3ζ chain (SEQ ID NO:4 or SEQ ID NO:8) and HSVTK suicide gene (SEQ ID NO:5 or SEQ ID NO:9 ), collectively referred to as anti-hCD20scFv-CD28z-CD3ζ-HSVTK sequence (such as figure 1 shown), the two ends of the sequence introduced the coding sequence of XbaI and SalI restriction endonuclease cutting sites. In the present invention, the full-length sequence of anti-hCD20scFv-CD28z-CD3ζ-HSVTK is artificially synthesized, and BGI Gene Co., Ltd. is commissioned to synthesize it for a fee. The third-generation lentiviral vector pLenti-CMV is selected, an...

Embodiment 2

[0054] Example 2: Viral packaging comprising a CAR structure

[0055] Use 293FT as packaging cells, and when they reach 90% confluence, replace with serum-free DMEM medium, and use Lipofectamine3000 as the transfection reagent. For a six-well plate, the amount of plasmids used for transfection is respectively, vsv-G0.37μg +tat 0.19μg+rev 0.19μg+gag 0.19μg+vector(pLenti-CMV-anti-hCD20scFv-CD28z-CD3ζ-HSVTK) 2μg, operate according to the instructions of Lipofectamine3000, after preparing the transfection solution, add it to the serum-free medium for culture In the 293FT, after 6 hours, replace it with 10% fetal bovine serum DMEM complete medium, continue to culture for 48-72 hours, collect the virus liquid, filter and centrifuge at 90,000gx for 2 hours, add 500μl PBS / tube, and freeze in - Standby at 80°C.

Embodiment 3

[0056] Example 3: Preparation of anti-hCD20-CAR-T (CAR20-T)

[0057] Using a 50ml syringe, collect 50ml of peripheral blood from a lymphoma patient at one time, dilute it with PBS at a rate of 1:2, and slowly add it to the Ficoll separation solution at a ratio of 2:1. Centrifuge at 1500rpm for 10 minutes at room temperature, it can be seen that it is divided into three layers, and the white layer in the middle is lymphocytes; gently suck the lymphocyte layer into 6ml~8mlRPMI1640 to elute; centrifuge at 1500rpm for 10 minutes; remove the supernatant, add 2mlRPMI1640, and count Press 0.5x 10 6 / ml added to OpTmizer TM In T-cell Expansion SFM medium, cultivate overnight. Mix the virus liquid obtained in the previous step with MOI 5, polybrene 10 μg / ml / , 1000 IU / ml recombinant human interleukin 2 (rhIL-2) and add it to T cells after overnight culture, the cell concentration is 0.5×10 6 / ml, after culturing overnight, replace with OpTmizer containing 1000IU / ml rhIL-2 TM T-cell ...

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Abstract

The invention relates to a chimeric antigen receptor (CAR) immune cell provided with a safety switch as well as a preparation method and an application of the CAR immune cell. The CAR immune cell carrying the safety mechanism (the safety switch) comprises a CAR coding nucleotide sequence, wherein the structure of the nucleotide sequence comprises a receptor structural domain for recognizing tumor-specific antigen or tumor-associated antigen, a transmembrane-stimulation structural domain, a CD3[zeta] stimulating signal transduction region and a suicide gene region. The CAR immune cell can be obtained as different immunological effect cells are amplified and a CAR sequence carrying a suicide mechanism is transduced; corresponding antigens of tumor cells are recognized by virtue of CAR; and the CAR immune cell can generate a specific killing effect on the tumor cells. The CAR immune cell, when used, is infused in a gradient mode and dynamic change in related cell factor levels is monitored; in case of need, a suicide gene can be started by virtue of drugs to scavenge the immunological effect cells, so that optimal balance between safety and a curative effect is achieved; therefore, the safety of the technology applied to the treatment of tumors in the clinical field is guaranteed to the greatest extent.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to the main structure of a gene-modified chimeric antigen receptor (CAR) immune effector cell carrying a safety mechanism, its preparation method and application. The nucleic acid sequence of the CAR cell carrying a safety mechanism encodes a CAR with a safety switch, and the nucleic acid sequence structure includes a chimeric antigen receptor domain that recognizes a tumor-specific antigen or a tumor-associated antigen, a transmembrane-stimulatory domain, and a CD3ζ Stimulates signal transduction regions, integrated suicide gene regions. Background technique [0002] Surgery, radiotherapy, chemotherapy, and palliative care, as the traditional treatment methods for malignant tumors, have always occupied a dominant position in anti-tumor therapy. In the past ten years, with the continuous deepening of the research on the internal mechanism of tumor development and its external mic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10A61K48/00A61K35/17A61P35/00A61P35/02
Inventor 袁顺宗刘兵苏航李庆虹孙磊廖辉阳
Owner AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI
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