A chimeric antigen receptor immune cell and its preparation method and application

A chimeric antigen receptor and immune cell technology, applied in the field of genetic engineering, can solve the problem of γδT cells not being treated with coronavirus infectious diseases, and achieve the effect of avoiding off-target effects, being infected by viruses, and preventing transformation

Active Publication Date: 2021-05-07
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, γδT cells have not been used in the treatment of coronavirus infectious diseases

Method used

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  • A chimeric antigen receptor immune cell and its preparation method and application
  • A chimeric antigen receptor immune cell and its preparation method and application
  • A chimeric antigen receptor immune cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Design of Chimeric Antigen Receptor

[0083] In this example, a chimeric antigen receptor ACE2-CAR with the 18-615 amino acids of human angiotensin-converting enzyme 2 (ACE2) as the antigen-binding domain was constructed, and on the basis of ACE2-CAR, a human Secreted angiotensin-converting enzyme 2 (human secreted ACE2, sACE2) functional chimeric antigen receptor ACE2-CAR-sACE2, the schematic diagram of the two chimeric antigen receptors is shown in Figure 2A , Figure 2B and image 3 As shown, ACE2-CAR includes a signal peptide (Leader), an antigen-binding domain (tACE2) of ACE2 extracellularly recognizing S protein, a hinge region (Hinge) and a transmembrane region (TM) of CD8α, a costimulatory domain of 4-1BB and CD3ζ signaling domain, ACE2-CAR-sACE2 includes signal peptide (Leader), ACE2 extracellular recognition S protein antigen binding domain (tACE2), CD8α hinge region (Hinge) and transmembrane region (TM), 4-1BB co- Stimulatory domain, CD3ζ signal...

Embodiment 2

[0085] Example 2 lentiviral packaging

[0086] The coding genes of ACE2-CAR and ACE2-CAR-sACE2 were respectively constructed into lentiviral vectors, and the four-plasmid system was used to package the two lentiviral vectors constructed by lentiviruses, and the steps were as follows;

[0087] Mix the helper plasmids gag / pol, Rev and VSV-G with one of the two lentiviral vectors in proportion, with a total mass of 10 μg, add it to a certain volume of serum-free DMEM, mix well and let it stand for 15 minutes; add the above mixture to In a cell culture flask with 293T cells, mix gently and store at 37°C, 5% CO 2 Cultivate in the cell incubator for 6 hours; after 6 hours, replace the fresh medium, continue to cultivate, and add 10mM sodium butyrate solution; after 72 hours, collect the lentivirus culture supernatant for purification and detection.

Embodiment 3

[0088] Example 3 Preparation of CAR-γδT cells

[0089] (1) Separation of PBMCs

[0090] Collect 50mL of peripheral blood; add 15mL of lymphocyte separation medium to two 50mL sterilized centrifuge tubes in the ultra-clean workbench, slowly inject 25-30mL of peripheral blood into the centrifuge tubes containing lymphocyte separation Centrifuge the centrifuge tube at 700g for 20 minutes at room temperature, increase the speed by 1, and decrease the speed by 2. If the blood is stored for more than 2 hours, increase the centrifugation time to 30 minutes;

[0091] After centrifugation, the blood is divided into 4 layers, which are composed of plasma (upper layer), mononuclear cells between plasma and separation liquid (2nd layer), separation liquid (3rd layer) and red blood cells (bottom layer). Collect the mononuclear cells into a new centrifuge tube, add 20mL PBS to dilute the cell suspension, centrifuge at 500g for 10min; remove the supernatant, add 20mL PBS to dilute the cell ...

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Abstract

The invention provides a chimeric antigen receptor immune cell and a preparation method and application thereof. The expressed CAR molecule includes a signal peptide, a binding receptor of the coronavirus spike glycoprotein, a hinge region, a transmembrane domain, and a costimulatory structure. domain and signaling domain; the CAR molecule also includes secretory ACE2, which is connected to the carboxyl terminus of the signaling domain through a linking peptide; the immune cells expressing the CAR molecule have the ability to target and clear the cells infected by the new coronavirus, and Release secreted ACE2 molecules to competitively bind to the viral S protein to achieve allogeneic reinfusion to treat people infected with COVID-19 and prevent the transformation from mild to critically ill. It also has universal curative effect on other viruses bound by ACE2 receptors, and has strategic reserve value It provides new methods and ideas for the treatment of many major human diseases such as tumors and immune diseases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a chimeric antigen receptor immune cell and its preparation method and application. Background technique [0002] Recently, the novel coronavirus (SARS-CoV-2) has ravaged the world, and the resulting coronavirus disease 2019 (COVID-19) has posed a huge threat and challenge to global public health. The virus was originally named 2019-nCoV by the World Health Organization (WHO), and is related to the previously identified human coronaviruses severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS) that can cause human disease. ) coronavirus (MERS-CoV), the novel coronavirus (SARS-CoV-2) may cause life-threatening disease and has pandemic potential. [0003] Coronavirus infection begins with binding to host cell surface receptors. Both SARS-CoV and MERS-CoV use the spike glycoprotein (S protein) on the surface of the viru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61K39/215A61P31/14
CPCA61K39/12A61K2039/5158A61P31/14C07K14/7051C07K16/10C07K2319/02C07K2319/03C12N5/0636C12N5/0646C12N15/86C12N2510/02C12N2740/15043C12N2770/20034
Inventor 李光超罗敏曾剑华周兆吴丽琴
Owner GUANGZHOU BIO GENE TECH CO LTD
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