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Construction method and application of NKG2D-ACE2 CAR-NK cell secreting super IL 15

A cell and extracellular segment technology, applied in DNA/RNA fragments, genetically modified cells, cells modified by introducing foreign genetic material, etc., can solve the problem of no effective removal of SARS-CoV-2 virus, etc., to achieve safety good effect

Pending Publication Date: 2020-07-28
温州启星生物技术有限公司
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  • Abstract
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Problems solved by technology

[0002] At present, there is no specific drug / therapy for clearing the SARS-CoV-2 virus, especially for severe COVID-19.

Method used

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  • Construction method and application of NKG2D-ACE2 CAR-NK cell secreting super IL 15
  • Construction method and application of NKG2D-ACE2 CAR-NK cell secreting super IL 15
  • Construction method and application of NKG2D-ACE2 CAR-NK cell secreting super IL 15

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Experimental program
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Effect test

Embodiment

[0023] Main experimental materials:

[0024] NdeI-HF, MluI-HF (NEB Company), Seamless Cloning Enzyme (He Yuanbio), High Fidelity Prime GXL STAR Enzyme (TAKARA Company), TransStbl3 Competent Cells (Quanshijin Biotechnology Co., Ltd.), Plasmid Mini Kit I (OMEGA), Plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sunny Biological Co., Ltd.), yeast powder, peptone, EDTA, NaOH (Shanghai Sangong Biological Engineering Co., Ltd.), primers (Jiangsu Jinweizhi Biotechnology Co., Ltd.).

[0025] 1. Construction of recombinant plasmids

[0026] ① Construction of Plenti-NKG2D-ACE2-CAR was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. NdeI-HF and MluI-HF were used to double-enzyme digest the plenti-EF1a--MCS vector and the synthesized NKG2D-ACE2CAR gene, so that both There are NdeI and MluI cohesive ends, the reaction conditions are 37°C for 3h, 65°C for 20min, and the enzyme digestion system is shown...

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Abstract

The invention discloses a construction method and application of an NKG2D-ACE2 CAR-NK cell secreting super IL 15. An expression vector mainly comprises an NKG2D extracellular segment, an ACE2 extracellular segment, 4-1BB, CD3[zeta] and IL15R[alpha]-IL15 thawing protein. NKG2D is an activation receptor of the NK cell and is used for identifying ligands (MICA, MICB, ULBP1-6) on the surfaces of virusinfected cells or tumor cells; ACE2 is a receptor of SARS-CoV-2 and is combined with S protein of a virus envelope; the NK cell modified by CAR is proved to be capable of breaking through the limitation of an inhibitory receptor to activate the NK cell, so that the specific killing effect of the NK cell on target cells is enhanced, and no serious toxic or side effect exists; and the activity of an IL15 super agonist is increased by 20 times compared with that of natural IL15 in the body, and the pharmacokinetic property is improved, so that the CAR-NK cell has enhanced durability and target cell killing activity. Therefore, the ACE2 and the NKG2D respectively target the S protein of the SARS-CoV-2 virus and NKG2DL on the surfaces of the infected cells, and the powerful synergistic effectof the super IL15 is achieved, so that the NKG2D-ACE2 CAR-NK cell can specifically remove SARS-CoV-2 virus particles and the infected cells, and a safe and effective cell therapy is provided for COVID-19.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and specifically relates to the construction of NKG2D-ACE2 CAR NK cells and their application in the prevention and treatment of COVID-19. Background technique [0002] At present, there is no specific drug / therapy for clearing the SARS-CoV-2 virus, especially for severe COVID-19. In view of the clinical observation that there is a very clear correlation between leucopenia and lymphopenia and the deterioration of the disease and even death, the current diagnosis and treatment plan also emphasizes the use of some immune enhancers in order to improve the immunity of severe and critically ill patients. To achieve the purpose of effectively controlling the replication of the SARS-CoV-2 virus. [0003] CAR-NK cells, also known as upgraded NK cells, are modified by genetic engineering to express chimeric antigen receptors (CAR), which will recognize antibodies (or Receptors) are connected to ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10
CPCC07K14/7051C07K14/7056C07K14/7155C07K14/5443C12N5/0646C12N9/485C12Y304/17023C12N2510/00C07K2319/33
Inventor 高基民魏成赵爱段德明
Owner 温州启星生物技术有限公司
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