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Fully-humanized anti-neocoronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application

A monoclonal antibody, fully human-sourced technology, applied in the field of microbiology and immunology, can solve the problems of Beta strain virus loss of neutralization activity, loss of neutralization activity, etc., and achieve broad-spectrum high-efficiency neutralization activity and good stability Effect

Active Publication Date: 2021-12-03
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the continuous evolution and mutation of SARS-CoV-2, most of the monoclonal antibodies under development have lost their neutralizing activity against one or more mutant strains. Among them, LY-CoV555 and LY-CoV016 developed by Lilly, regeneration REGN10933 developed by Yuan loses neutralizing activity against Beta strain virus

Method used

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  • Fully-humanized anti-neocoronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application
  • Fully-humanized anti-neocoronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application
  • Fully-humanized anti-neocoronavirus broad-spectrum high-neutralization-activity monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Example 1 Screening and Preparation of Human Anti-SARS-CoV-2 Monoclonal Antibody

[0038] 1. Blood Sample Collection

[0039] After obtaining informed consent, 20 mL of blood samples were collected 14 days after the second immunization of the recombinant new coronavirus vaccine vaccinators for subsequent experiments.

[0040] 2. Sorting Memory B Cells by Flow Cytometry

[0041] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:

[0042] 1) Take fresh anticoagulated whole blood and anticoagulate with EDTA.

[0043] 2) Add the same volume of separation liquid as the blood sample into the centrifuge tube, spread the blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear.

[0044] 3) Trim, room temperature, horizontal rotor 800g, acceleration and deceleration speed 3, centrifuge for 30min.

[0045] 4) After centrifugation, the...

Embodiment 2

[0120] Example 2. Antibody ZWD12 recognition epitope analysis

[0121]1) Coating: Take the recombinant SARS-CoV-2 S-ECD antigen, S1 antigen, RBD antigen and S2 antigen on the 96-well enzyme-linked plate one day before the experiment and dilute it with coating solution to a concentration of 2 μg / mL, and coat the enzyme-linked plate , 100 μL per well, and coated overnight at 4°C.

[0122] 2) Blocking: Wash 3 times with a plate washer (BIO-TEK, 405_LS) on the day of the experiment, add 100 μL of blocking solution to each well, and incubate at 37° C. for 1 hour.

[0123] 3) Sample incubation: wash the plate 3 times, add 100 μL of diluent to each well except the first well, dilute the antibody to 1 μg / mL in the first well, 4-fold serial dilution, 100 μL / well, set up three replicate wells for each antibody, Incubate at 37°C for 1 h.

[0124] 4) Secondary antibody incubation: wash the plate 3 times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1...

Embodiment 3

[0130] Example 3: Identification of cross-binding activity of antibody ZWD12

[0131] The cross-binding activity between ZWD12 and SARS-CoV-2 Variants of concern (Variants of concern) S protein was identified, the method was the same as above, and the results were as follows Figure 8 shown. ZWD12 specifically binds to the S-ECD protein of Alpha strain, Beta strain, Gamma strain, and Delta strain, and exhibits a dose-response relationship. The results showed that monoclonal antibody ZWD12 could cross-link the S-ECD protein of Alpha strain, Beta strain, Gamma strain and Delta strain.

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Abstract

The invention discloses an anti-SARS-CoV-2 fully-humanized monoclonal antibody ZWD12. The antibody is obtained through screening by a flow sorting-single cell PCR technology and has a unique CDR partition, and the antigen recognition epitope of the antibody is located in the RBD region of S1 protein. The EC50 of the antibody to a neocoronavirus wild type is 0.103 microgram / mL, the EC50 of a neutralized Beta strain is 0.069 microgram / mL, the EC50 of a neutralized Delta strain is 0.079 microgram / mL, the ZWD12 has broad-spectrum efficient neutralization activity to current main variants. The monoclonal antibody disclosed by the invention also has the advantages of being high in expression, fully humanized and good in stability, is suitable for industrial production and has great clinical application value for dealing with outbreak and prevalence caused by the current variants and variants possibly appearing in the future.

Description

technical field [0001] The invention discloses an antibody and belongs to the fields of microbiology and immunology. Background technique [0002] The pathogen of new coronary pneumonia is novel coronavirus-2 (SARS-CoV-2). SARS-CoV-2 belongs to the β-coronavirus genus of Coronaviridae. The length is about 30kb. The first 2 / 3 of the genome is the non-structural gene ORF1a / b, which mainly encodes enzymes related to viral replication (RNA-dependent RNA polymerase, RdRp), and the last 1 / 3 encodes four structural proteins in turn: spike protein (S), Envelope (E), Membrane (M) and Nucleocapsid (N). Among them, the S protein contains a virus receptor binding region, which can bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of human cells, mediate virus adsorption and entry into cells, and is a key protein for virus invasion of host susceptible cells. [0003] The virus will continuously mutate randomly during the transmission process, some of which wil...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/565C07K2317/52C07K2317/33C07K2317/76C07K2317/92C12N2800/107A61K2039/505Y02A50/30
Inventor 陈薇于长明迟象阳孙韩聪张冠英张军李建民范鹏飞王步森宰晓东张哲房婷付玲陈郑珊郝勐陈旖徐婧含
Owner ACADEMY OF MILITARY MEDICAL SCI
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