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Fully human monoclonal antibody for resisting novel coronavirus and application of fully human monoclonal antibody

A monoclonal antibody, coronavirus technology, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problems of limited plasma sources of recovered patients, the risk of disease transmission between batches, and the inability to popularize and apply, and achieve inhibition. The effect of virus infection on host cells, good druggability, high neutralization activity

Active Publication Date: 2022-06-24
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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  • Fully human monoclonal antibody for resisting novel coronavirus and application of fully human monoclonal antibody
  • Fully human monoclonal antibody for resisting novel coronavirus and application of fully human monoclonal antibody
  • Fully human monoclonal antibody for resisting novel coronavirus and application of fully human monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Screening and Preparation of Human Anti-SARS-CoV2 Monoclonal Antibody

[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 AviTag fusion protein biotinylation kit (Avidity, BirA500) was used for biotin labeling of probes. The reaction system was prepared according to the steps 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 intervals of 20 min until the molar ratio of SA-PE or SA-APC to biotin-labeled probe reached 1:1, and incubated at 4 °C with gentle shaking to prepare positive and negative probes labeled with fluorescein: Ag(-...

Embodiment 2

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

[0101] Antibody-antigen binding activity analysis using Gator label-free biomolecular analyzer

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

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

[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 by 1000 times, continue to perform gradient dilution according to 1:3, a total of 4 dilutions (36.2nM, 12.1 nM, 4.02 nM and 1.34 nM), take 200 μL of each dilution samp...

Embodiment 3

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

[0110] Analysis of Antibody Blocking Binding Ability of SARS-CoV2 Virus RBD and Receptor ACE2 Using Gator Non-labeled Biomolecular Analyzer

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

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

[0113] 3.3 During the detection process, each experiment was first equilibrated in the HBS-EP buffer of the probe card for 120 s, ...

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Abstract

The invention relates to the field of biological medicine, and discloses a fully human monoclonal antibody for resisting novel coronavirus and application of the fully human monoclonal antibody. A receptor binding domain (RBD) of virus S protein is used as a probe, flow cytometry is used for screening memory B lymphocytes of a novel coronavirus pneumonia rehabilitation patient, and the affinity constant of the prepared monoclonal antibody and the RBD is 4.48 nM. X-ray crystal diffraction is used for analyzing the three-dimensional structure of an antibody and RBD compound, it is found that binding epitopes of the antibody and a host cell receptor (ACE2) are partially overlapped, binding of the RBD and the ACE2 can be competitively blocked, the virus is prevented from infecting host cells, some genetic mutations of the novel coronavirus can be effectively coped with, and the novel coronavirus can be effectively prevented from being infected. The lowest half effective inhibition concentration of the compound for blocking the infection of the novel coronavirus on host cells can reach 20.93 ng / mL, and the compound has the advantages of safety, high efficiency and broad spectrum, and is expected to be used for diagnosing, treating and preventing novel coronavirus 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 novel coronavirus (SARS-CoV2), which can cause acute respiratory disease. The clinical features of COVID-19 include fever, dry cough and fatigue, respiratory failure, etc. SARS-CoV2 belongs to the genus Coronavirus of the Coronaviridae family, and is a class of enveloped single-stranded positive-sense RNA viruses. The viral gene code contains multiple structural proteins, such as Spike protein (S protein), envelope 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 the virus cell envelope, while N protein is involved i...

Claims

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Application Information

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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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