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

A monoclonal antibody and coronavirus technology, applied in antiviral agents, antiviral immunoglobulins, applications, etc., can solve the problems of limited plasma sources of recovered patients, difficulty in large-scale preparation, and risk of disease infection due to batch-to-batch differences

Active Publication Date: 2021-05-14
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

[0034] Example 1: Screening and preparation of fully human anti-new coronavirus monoclonal antibody

[0035] 1.1 Labeling of probes

[0036] The required SARS-CoV2-related specific positive and negative probes are conjugated with fluorescent dyes, and the process is as follows:

[0037] 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.

[0038] 1.1.2 Use a 30 kDa ultrafiltration tube to exchange the reaction product with 1× PBS (pH 7.4) to remove excess biotin.

[0039] 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 fluore...

Embodiment 2

[0075] Example 2: Analysis of the binding activity of monoclonal antibody YK-CoV-02 to RBD protein

[0076] Antibody-antigen binding activity analysis using Gator label-free biomolecular analyzer

[0077] 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).

[0078] 2.2 The biotinylated RBD was diluted 600 times, 3 μL of RBD was added to 2000 μl of HBS-EP buffer and mixed, and the diluted RBD was loaded into a 96-well sample plate at an amount of 200 μL per well.

[0079] 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), ta...

Embodiment 3

[0084] Example 3: Analysis of the ability of monoclonal antibody YK-CoV-02 to competitively block the binding of RBD to the receptor ACE2

[0085] Analysis of Antibody Blocking Binding Ability of SARS-CoV2 Virus RBD and Receptor ACE2 Using Gator Non-labeled Biomolecular Analyzer

[0086] 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.

[0087] 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 .

[0088] 3.3 During the detection process, each experiment was first equilibrated in the HBS-EP buffer of the probe card for 120 s, and the...

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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. An SARS-CoV2 receptor binding domain (RBD) is used as a probe, flow cytometry is used for screening B lymphocytes of a new coronary pneumonia rehabilitation patient, and the affinity constant of the prepared monoclonal antibody and the RBD is 4.03 nM. X-ray crystal diffraction is used for analyzing the three-dimensional structure of the antibody and RBD compound to find that binding epitopes of the antibody and a host cell receptor (ACE2) are 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 new coronavirus can be coped with. The median effective concentration (IC50) for blocking pseudoviruses (accumulated B.1. 1.7 mutant strain genes and D614G) from infecting host cells is 0.12 ng / mL, and the application prospect of diagnosing, treating and preventing the new coronal pneumonia is achieved.

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 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 216 countries and regions, 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 used for prevention and specific drugs for the treatment of novel coronavirus pneumonia are still in the stage of clinical trials or emergency use under specific conditions, and their safety and effectiveness need to be further verified. [0003] SARS-CoV2 belongs to the genus Coronavirus of the Coronaviridae family, and is a class of envelo...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14G01N33/577G01N33/569
CPCA61P31/14C07K16/10C07K2317/56C07K2317/76C07K2317/92G01N33/56983G01N33/577G01N2333/165G01N2469/10
Inventor 陈磊高腾森戴敏张冀王聪赵氏璧谢欣周翔宇王得荣陈莉周臣飞
Owner SUZHOU YUZHIBO BIOLOGICAL TECH CO LTD
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