A fully human monoclonal antibody against novel coronavirus and its application

A monoclonal antibody and coronavirus technology, applied in antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problems of limited plasma sources of recovered patients, the risk of disease infection due to batch-to-batch differences, and the inability to popularize and apply it to achieve inhibition Effect of virus infecting host cells, good druggability, high and high activity

Active Publication Date: 2022-08-09
SUZHOU YUZHIBO BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited source of plasma from recovered patients, it is difficult to prepare on a large scale, and there are factors such as batch-to-batch differences and risks of other disease infections, making this therapy unable to be widely used.

Method used

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  • A fully human monoclonal antibody against novel coronavirus and its application
  • A fully human monoclonal antibody against novel coronavirus and its application
  • A fully human monoclonal antibody against novel coronavirus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Screening and preparation of human anti-SARS-CoV2 monoclonal antibodies

[0065] 1.1 Labeling of probes

[0066] The required SARS-CoV2-related specific positive and negative probes are conjugated with fluorescent dyes, and the process is as follows:

[0067] 1.1.1 Use AviTag Fusion Protein Biotinylation Kit (Avidity, BirA500) for biotin labeling of the probe. The reaction system was prepared according to the procedure described in the manual and incubated at 30 °C for 30 min.

[0068] 1.1.2 Use a 30 kDa ultrafiltration tube to exchange the reaction product with 1× PBS (pH 7.4) to remove excess biotin.

[0069] 1.1.3 Gradually add 1 / 5 molar equivalent of streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC) to the biotin label at 20 min intervals Among the probes, until the molar ratio of SA-PE or SA-APC to biotin-labeled probe reaches 1:1, incubate at 4 °C with gentle shaking to prepare positive and negative probes labeled with fluor...

Embodiment 2

[0100] Example 2: Analysis of the binding activity of monoclonal antibody YK-CoV2-03 to RBD protein

[0101] Antibody and Antigen Binding Activity Analysis Using Gator Unlabeled Biomolecule Analyzer

[0102] 2.1. Using GNTi cells to express RBD, after purification by nickel column and molecular sieve in turn, biotinylated RBD according to the steps shown in the instructions of DSB-X™ Biotin Labeling Kit (Invitrogen™, D20655).

[0103] 2.2 Dilute the biotinylated RBD by 600 times, add 3 μL of RBD to 2000 μL of HBS-EP buffer and mix, and load the diluted RBD into a 96-well sample plate in an amount of 200 μL per well.

[0104] 2.3 Use HRV 3C protease to cut the monoclonal antibody into Fab fragments, use affinity chromatography and molecular sieve to purify the obtained antibody Fab, after diluting the antibody Fab 1000 times, continue to carry out gradient dilution according to 1:3, a total of 4 dilutions (36.2nM, 12.1nM, 4.02nM and 1.34nM), take 200 μL of each dilution sample...

Embodiment 3

[0109] Example 3: Analysis of the ability of monoclonal antibody YK-CoV2-03 to competitively block the binding of SARS-CoV2 virus RBD to receptor ACE2

[0110] Analysis of the binding ability of antibodies to block SARS-CoV2 virus RBD and receptor ACE2 using Gator non-labeled biomolecule analyzer

[0111] 3.1 Expi-CHO cells were used to express the receptor ACE2, and after purification by nickel column and molecular sieve in sequence, ACE2 was biotinylated according to the steps shown in the instructions of DSB-X™ Biotin Labeling Kit (Invitrogen™, D20655). Biotinylated ACE2 was diluted to 40 μg / mL at a molar concentration of 570 nM.

[0112] 3.2 Dilute RBD to 30 μg / mL with a molar concentration of 1000 nM, dilute the antibody to 0.2 mg / mL, and mix with RBD in a molar ratio of 1:1. The molar amount of antibody in the prepared mixture is greater than the molar amount of ACE2. .

[0113] 3.3 During the detection process, each experiment was first equilibrated in the HBS-EP buff...

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Abstract

The present invention involves the field of biomedical, and a whole -human source monoclonal antibody and its application of anti -new coronary virus are disclosed.Based on the receptor of the virus S protein as the probe, the memory B lymphocytes are used to screen the new coronary virus pneumonia patients with streaming cells, and the affinity constant of a single cloning antibody and RBD is 4.48 nm.The three -dimensional structure of the X -ray crystal diffraction antibody and the RBD complex found that the combination of antibodies and the host cell receptor (ACE2) of the host cells overlapped, which can competitively block the combination of RBD and ACE2, inhibit the virus infection of host cells.It can effectively respond to some genetic mutations in the new coronary virus, and half of the number of effective inhibitory concentrations that block the new coronary fake virus infection can reach a minimum of 20.93 ng / ml.Treatment and preventing new coronary virus pneumonia.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fully human monoclonal antibody against novel coronavirus and its application. Background technique [0002] The causative agent of novel coronavirus pneumonia (COVID-19) is the novel coronavirus (SARS-CoV2), which can cause acute respiratory disease. The clinical features of COVID-19 include fever, dry cough and fatigue, and respiratory failure. SARS-CoV2 belongs to the family Coronaviridae and belongs to the genus Coronavirus, which is a class of enveloped single-stranded positive-stranded RNA viruses. The viral gene encoding contains multiple structural proteins, such as spike protein (Spike protein, S protein), membrane protein (Envelope protein, E protein), membrane protein (Membrane glycoprotein, M protein) and nucleocapsid protein (Nucleocapsid protein, N protein), etc.; among them, S protein, M protein and E protein are involved in the formation of viral cell enve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
CPCC07K16/10A61P31/14G01N33/577G01N33/56983C07K2317/565C07K2317/567C07K2317/51C07K2317/515C07K2317/76C07K2317/92G01N2333/165G01N2469/10A61K2039/505
Inventor 陈磊高滕森殷建国
Owner SUZHOU YUZHIBO BIOLOGICAL TECH CO LTD
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