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Armed chimeric antigen receptor cell targeting coronavirus SPIKE, preparation methods and applications

A technology of chimeric antigen receptors and viruses, which can be applied to viruses/bacteriophages, genetically modified cells, and methods based on microorganisms, and can solve the problem of no specific and effective therapeutic drugs for diseases

Pending Publication Date: 2020-09-18
NANJING KAEDI BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no specific and effective treatment for the disease caused by the new coronavirus

Method used

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  • Armed chimeric antigen receptor cell targeting coronavirus SPIKE, preparation methods and applications
  • Armed chimeric antigen receptor cell targeting coronavirus SPIKE, preparation methods and applications
  • Armed chimeric antigen receptor cell targeting coronavirus SPIKE, preparation methods and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1: Preparation of specific knockout SARS-CoV-2 virus RNA polymerase construction targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor expression plasmid (KD-2019 lentiviral vector)

[0150] The overall design is as follows:

[0151] 1. Determination of the amino acid sequence of SARS-CoV-2 virus SPIKE-armed chimeric antigen receptor by specifically knocking out SARS-CoV-2 virus RNA polymerase

[0152] First, the full-length amino acid sequence of human ACE2 (NP_068576.1) and the full-length amino acid sequence of SPIKE protein (YP_009724390.1) were searched from the Genbank database of the National Library of Medicine (NCBI).

[0153] Secondly, construct specific knockout SARS-CoV-2 virus RNA polymerase to construct targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor, that is to determine the amino acid sequence of armed chimeric antigen receptor molecule:

[0154] From the amino terminal to the carboxyl terminal, the amino acid sequenc...

Embodiment 2

[0170] Example 2: Preparation of the virus liquid (KD-2019 virus liquid) of the lentiviral vector

[0171] The recombinant plasmid (KD-2019CoV lentiviral vector) and packaging vector targeting the SARS-CoV-2 virus SPIKE arming chimeric antigen receptor were constructed by expressing specifically knocking out the SARS-CoV-2 virus RNA polymerase obtained in Example 1 psPAX2 and VSVG, according to the ratio of 10:8:5, with Lipofectamine TM 6000 transfection reagent (purchased from ThermoFisher, product model 11668019) co-transfected 293T cells (see ThermoFisher transfection manual for specific transfection operation process), and replaced with complete medium 6 hours after transfection (purchased from Life Technologies , the product model is 11995-065), after 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain SARS-CoV carrying expression-sp...

Embodiment 3

[0172] Example 3: Isolation and culture of T cells

[0173] Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation; paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information for Human T-Activator CD3 / CD28) to enrich CD3+ T cells, specifically, dilute peripheral blood mononuclear cells to a concentration of (10-30)×10 6 A single cell / ml, and then the magnetic beads and cells were mixed in a culture dish at a ratio of 3:1, and co-incubated at room temperature for 2-3 hours. ) to enrich CD3+ T cells. Finally, the enriched CD3+T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizer TM In T-CellExpansion SFM), the cell solubility was adjusted to 1×10 6 pcs / ml, finally at 37°C, 5% CO 2 Cultivate in the incubator for 2 days, see the test results Figure 4 . ...

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Abstract

The invention relates to a coding amino acid sequence of an armed chimeric antigen receptor capable of specifically knocking out and targeting the SARS-CoV-2 virus SPIKE protein constructed by SARS-CoV-2 virus RNA polymerase, an immune response cell modified by the amino acid sequence, preparation methods and applications. The recombinant vector of the armed chimeric antigen receptor targeting theSARS-CoV-2 virus SPIKE can be constructed by knocking out the SARS-CoV-2 virus RNA polymerase, and a method using the recombinant vector to modify the immune response cell is also constructed; and the obtained novel functional immune response cell can effectively target and attack various viruses. The preparation method of the immune response cell modified by the armed chimeric antigen receptor targeting the SARS-CoV-2 virus SPIKE is simple in step; and the obtained immune response cell targeting the virus SPIKE and modified by virus replication-dependent RNA polymerase has obvious killing effects on coronavirus.

Description

technical field [0001] The invention belongs to the technical field of immunotherapy biomedicine, and relates to a method of specifically knocking out SARS-CoV-2 virus RNA polymerase to construct an armed chimeric antigen receptor targeting the SPIKE protein of SARS-CoV-2 virus to antagonize cytokine storm Amino acid coding sequence, immune response cells modified therefor, their preparation method and application in medicine preparation. Background technique [0002] Coronaviruses are a large family of viruses known to cause colds as well as more serious illnesses such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has never been found in humans before. Common signs after a person is infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrom...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N7/01C12N15/867A61K39/215A61K35/17A61P31/14C12R1/93
CPCC12N9/485C12Y304/17023C07K14/7051C07K16/2866C12N5/0636C12N5/0646C12N15/86C12N7/00A61K39/12A61K35/17A61P31/14C07K2319/03C07K2319/02C07K2319/33C07K2317/622C12N2510/00C12N2740/15043C12N2740/15021C12N2770/20034A61K2039/5158
Inventor 代红久徐慧朱靓婧
Owner NANJING KAEDI BIOTECH INC
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