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

A coronavirus and monoclonal technology, applied in the direction of antiviral agents, antiviral immunoglobulins, antibodies, etc., can solve the problems of limited plasma sources, difficulty in large-scale preparation, and inability to be popularized and applied on a large scale

Active Publication Date: 2021-09-24
SUZHOU YUZHIBO BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of convalescent plasma therapy has shown good therapeutic effects in the treatment of critically ill patients. However, due to limited plasma sources, difficulty in large-scale preparation, large batch-to-batch differences, and the risk of other disease infections, this therapy cannot be used on a large scale. Promoted application of

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Screening and preparation of fully human anti-SARS-CoV-2 monoclonal antibody

[0085] 1.1 Labeling of protein probes

[0086] Coupling the required SARS-CoV-2-related specific probes to fluorescent dyes, the process is as follows:

[0087] 1.1.1 Prepare the reaction system according to the instructions of the AviTag Fusion Protein Biotinylation Kit (BirA500, Avidity). Incubate at 30°C for 30 min.

[0088] 1.1.2 Use a 30 kDa ultrafiltration tube to exchange the reaction product with 1× PBS (pH 7.4) to remove excess biotin.

[0089] 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 in the antigen-specific probe until the molar ratio of SA-PE or SA-APC:biotin-labeled antigen-specific probe reaches 1:1. Incubate with gentle shaking at 4 °C. Fluorescein-labeled probes were thus prepared: Ag(-)-SA-APC and Ag(+)-SA-PE, respectively.

[0090] 1.2 Sepa...

Embodiment 2

[0123] Example 2 Analysis of the binding activity of YK-CoV2-01 antibody to RBD protein

[0124] Antibody-antigen binding activity analysis using Gator label-free biomolecular analyzer

[0125] 2.1. Expression of RBD-chimera using GNTI cells. After being purified by nickel column and molecular sieve sequentially, the RBD chimera was biotinylated according to the steps indicated in the manual of DSB-X™ Biotin Protein Labeling Kit (Invitrogen™).

[0126] 2.2 The biotinylated RBD chimera was diluted 600 times, and 3 μL of the RBD chimera was added to 2000ul HBSEP buffer and mixed. The diluted RBD was loaded into a 96-well sample plate at 200 μL per well.

[0127] 2.3 Use HRV3C enzyme to cut the antibody to be detected into Fab fragments, and use affinity chromatography and molecular sieve to purify the obtained antibody Fab. After diluting the antibody Fab by 1000 times, continue to perform serial dilution according to 1:3, a total of 4 dilutions (36.2nM, 12.1 nM, 4.02 nM and ...

Embodiment 3

[0132] Example 3 Analysis of YK-CoV2-01 Antibody Competitively Blocking the Binding Ability of SARS-CoV2 RBD and Receptor ACE2

[0133] Analysis of Antibody Blocking Binding Ability of SARS-CoV-2 Viral RBD Chimera and Receptor ACE2 Using Gator Non-Labeled Biomolecular Analyzer

[0134] 3.1 Using Expi-CHO cells to express the receptor ACE2. After purification by nickel column and molecular sieve in sequence, ACE2 was biotinylated according to the steps indicated in the manual of DSB-X™ Biotin Protein Labeling Kit (Invitrogen™). The biotinylated ACE2 receptor was diluted to 40ug / ml, and the molar concentration was 570nM.

[0135] 3.2 Dilute RBD to 30 ug / ml, molar concentration 1000 nM. The antibody was diluted to 0.2mg / ml and mixed 1:1 with the RBD chimera. The molar quantity of the antibody in the prepared mixture is greater than the molar quantity of the receptor ACE2.

[0136] 3.3 During the detection process, each experiment is first equilibrated in the HBSEP buffer of t...

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Abstract

The invention relates to the field of biomedicine, and discloses a fully human monoclonal neutralizing antibody against novel coronavirus and its application. The B-type lymphocytes were screened from the blood of recovered patients with new coronary pneumonia, and the affinity constant of the obtained antibody to the virus receptor binding domain (RBD) was 1.19 nM. Using X-ray crystallography to analyze the three-dimensional structure of the antibody-RBD complex, it was found that the binding epitope of the antibody highly overlaps with the binding epitope of the host cell receptor (ACE2), which can competitively block the binding of RBD and ACE2, and inhibit its infection of the host cell. The antibody can respond to certain genetic mutations of the virus, and the half effective concentration of blocking pseudovirus (accumulated B.1.1.7 mutant gene and D614G) from infecting host cells is 0.57 ng / mL, which has the advantages of safety, high efficiency and broad spectrum , is expected to be used as a neutralizing antibody drug for the diagnosis, treatment and prevention of new coronary pneumonia.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a fully human monoclonal neutralizing antibody against novel coronavirus and its application. Background technique [0002] The causative agent of novel coronavirus pneumonia (CoVID-19) is a novel coronavirus (SARS-CoV2), which can cause severe acute respiratory disease. According to the report of the World Health Organization, the epidemic has spread to all parts of the world, causing a large number of casualties. Clinical features of COVID-19 include fever, dry cough and fatigue, respiratory failure, and even death. So far, most of the vaccines and therapeutic drugs against COVID-19 are still in clinical trials and initial emergency trials, and their safety and effectiveness need further verification. [0003] SARS-CoV2 belongs to the genus Coronavirus of the Coronaviridae family, and is a class of enveloped single-stranded positive-sense RNA viruses. The new coronavirus gene enco...

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
CPCA61P31/14C07K16/10C07K2317/24C07K2317/56C07K2317/565C07K2317/567C07K2317/76C07K2317/92G01N33/56983G01N33/577G01N2333/165G01N2469/10
Inventor 陈磊高腾森戴敏殷建国赵氏璧王聪谢欣张冀周臣飞陈莉周翔宇王得荣
Owner SUZHOU YUZHIBO BIOLOGICAL TECH CO LTD
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