Neutralizing antibody for novel coronavirus SARS-CoV-2 and application of neutralizing antibody

A coronavirus, sars-cov-2 technology, applied in the field of biomedicine, can solve problems such as non-neutralization

Active Publication Date: 2021-09-17
SHANGHAI SYMRAY BIOPHARMA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that, unlike SARS-CoV, several key amino acids in the RBD region of the Spike protein of SARS-CoV-2 new coronavirus have mutations that bind to ACE2, resulting in the neutralizing antibodies against SARS-CoV developed in the past may not have the neutralizing effect. and the role of the virus

Method used

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  • Neutralizing antibody for novel coronavirus SARS-CoV-2 and application of neutralizing antibody
  • Neutralizing antibody for novel coronavirus SARS-CoV-2 and application of neutralizing antibody
  • Neutralizing antibody for novel coronavirus SARS-CoV-2 and application of neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1 - Screening of neutralizing antibodies

[0153] This embodiment is the screening of fully humanized scFv single chain antibody, and its steps include:

[0154] (1) Establishment of a fully human phage antibody library;

[0155] The peripheral blood of patients who had been infected with 2019-nCOV virus and recovered was collected, and the serum titer was determined. Select the sample with high titer to isolate lymphocytes, extract the total RNA of the cells, synthesize cDNA, use specific antibody heavy chain primers and light chain primers, amplify the variable region gene of the antibody, clone it into the phage display vector, and transfect into the host cell. Through NGS sequencing, the diversity and effectiveness of the antibody library were evaluated by quality control indicators such as library capacity, clone positive rate, heavy and light chain pairing, CDR3 distribution, and phage display rate.

[0156] (2) Phage library screening: use biotinylated...

Embodiment 2

[0161] Expression and purification of embodiment 2-scFv

[0162] In this embodiment, the 45 scFv single-chain antibodies screened in Example 1 were expressed and purified, which included the following steps:

[0163] Take the Escherichia coli TG1 bacterium liquid after shaking slightly, inoculate according to the ratio of 1:100, 37 ℃, 220rpm culture. When the OD is around 0.5, centrifuge at 8000rpm at room temperature for 5 minutes to obtain a bacterial liquid precipitate, induce with IPTG, culture at 30°C for about 12 hours, collect the expression supernatant for purification. The purification steps are as follows:

[0164] 1) Gently invert the bottle several times to mix the medium evenly;

[0165] 2) Draw a certain amount of medium and add it to the column, the column height is about 3-4 cm, and seal it when the medium settles to the bottom;

[0166] 3) Drain the storage solution, add 10mL equilibration buffer to equilibrate the chromatographic medium;

[0167] 4) Use a...

Embodiment 3-E

[0175] Embodiment 3-ELISA detects the binding activity of scFv

[0176] In this example, antibodies HTS0422, HTS0433, HTS0446 and HTS0483 were used as examples to detect the binding ability of purified scFv to Spike1-RBD antigen by ELISA. Specific steps are as follows:

[0177] 1) 384-well plate was coated with antigen, and 25 μL of 1 μg / mL Spike1-RBD antigen was added to each well, sealed, and coated overnight at 4°C;

[0178] 2) Discard the supernatant, wash 3 times with 100 μL Wash Buffer per well;

[0179] 3) 25 μL of blocking solution per well, block at room temperature for 1 hour, discard the liquid from the blocked ELISA plate, and wash 3 times with 100 μL of WashBuffer per well;

[0180] 4) Dilute the scFv antibody, the highest concentration is 200 μg / ml, four-fold serial dilution, a total of 8 points;

[0181] 5) Add 25 μL / well of the diluted antibody into appropriate wells and incubate at room temperature for 2 hours;

[0182] 6) Discard the supernatant, dry the ...

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Abstract

The invention discloses an antibody. The antibody can be bound to an RBD region of a S1 subunit of a Spike protein of a novel coronavirus SARS-CoV-2, so that the activity of binding between a host cell membrane surface receptor ACE2 and the Spike protein is blocked; the antibody is a neutralizing fully-human-derived antibody; and the antibody comprises a heavy chain VH and a light chain VL, and both the VH and the VL have variable regions CDR1, CDR2 and CDR3. The invention also relates to related application of the antibody, in particular to preparation of a complete IgG antibody and an affinity-matured antibody and application of the corresponding antibody in treatment and/or prevention of the novel coronavirus SARS-CoV-2. The fully-human-derived scFv single-chain antibody obtained by screening and the IgG antibody thereof can recognize and bind the RBD region of the Spike protein, can effectively block the activity of binding between ACE2 and the Spike protein, are non-toxic to cells, and can remarkably inhibit a cytopathic effect caused by virus infection, and after the affinity of the antibody is mature, both the affinity of the antibody and the blocking effect on the Spike protein and the ACE2 are obviously enhanced.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a novel coronavirus SARS-CoV-2 neutralizing antibody and its application. Background technique [0002] The average incubation period of novel coronavirus (SARS-CoV-2) pneumonia is 3-7 days, and the upper respiratory symptoms of infection are not prominent. Some patients with novel coronavirus infection only show low-grade fever, mild fatigue, etc., and no pneumonia. In severe and critically ill patients, moderate to low fever, or even no obvious fever can be manifested during the course of the disease. These non-specific clinical manifestations have brought greater challenges to epidemic prevention and control. Effective vaccines and therapeutic drugs are critical to controlling the spread of the virus and improving patient outcomes. However, as of now, there is still no vaccine against the coronavirus. In terms of drug treatment, broad-spectrum interferon and lopinavir / ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/569
CPCC07K16/10A61P31/14G01N33/56983C07K2317/76C07K2317/622C07K2317/56C07K2317/565C07K2317/92A61K2039/505G01N2333/165
Inventor 赵文祥刘明耀魏海涛褚敏晁瑞华尚玉栓王平李亚男夏永洋
Owner SHANGHAI SYMRAY BIOPHARMA CO LTD
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