Fully humanized broad-spectrum neutralizing antibody ZW2G10 for resisting new coronavirus and application of fully humanized broad-spectrum neutralizing antibody ZW2G10

A fully human, monoclonal antibody technology, applied in the fields of microbiology and immunology, can solve problems such as loss of neutralizing activity

Active Publication Date: 2022-02-11
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 Eli Lilly, regen

Method used

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  • Fully humanized broad-spectrum neutralizing antibody ZW2G10 for resisting new coronavirus and application of fully humanized broad-spectrum neutralizing antibody ZW2G10
  • Fully humanized broad-spectrum neutralizing antibody ZW2G10 for resisting new coronavirus and application of fully humanized broad-spectrum neutralizing antibody ZW2G10
  • Fully humanized broad-spectrum neutralizing antibody ZW2G10 for resisting new coronavirus and application of fully humanized broad-spectrum neutralizing antibody ZW2G10

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Screening and preparation of human anti-SARS-CoV-2 monoclonal antibodies.

[0037] 1. Collection of blood samples.

[0038] After obtaining informed consent, 20 mL of blood samples were collected from the recipients immunized with the recombinant novel coronavirus vaccine 14 days after the second immunization for subsequent experiments.

[0039] 2. Sorting memory B cells by flow cytometry.

[0040] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:

[0041] 1) Take fresh anticoagulated whole blood and anticoagulate with EDTA.

[0042] 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.

[0043] 3) Trim, room temperature, horizontal rotor 800 g, acceleration and deceleration speed 3, centrifuge for 30 min.

[...

Embodiment 2

[0119] Example 2. Analysis of the epitope recognized by antibody ZW2G10.

[0120] 1) Coating: Take the recombinant SARS-CoV-2 WT S-ECD antigen, S1 antigen, RBD antigen and S2 antigen on the 96-well enzyme-linked plate one day before the experiment and dilute to a concentration of 2 μg / mL with coating solution, and coat ELISA plate, 100 μL per well, coated overnight at 4°C.

[0121] 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.

[0122] 3) Sample incubation: wash the plate 3 times, add 100 μL 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 three for each antibody Duplicate the wells and incubate at 37°C for 1 h.

[0123] 4) Secondary antibody incubation: wash the plate 3 times, dilute the HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10000...

Embodiment 3

[0129] Example 3: Identification of cross-binding activity of antibody ZW2G10.

[0130] The cross-binding activity between ZW2G10 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 7 shown. ZW2G10 specifically binds to the S-ECD protein of Alpha strain, Beta strain, Gamma strain, Delta strain, and Omicron strain, and exhibits a dose-response relationship. The results showed that monoclonal antibody ZW2G10 could cross-link the S-ECD protein of Alpha strain, Beta strain, Gamma strain, Delta strain and Omicron strain.

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Abstract

The invention discloses an anti-SARS-CoV-2 fully humanized monoclonal antibody ZW2G10, the antibody 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 for neutralizing new coronavirus wild type pseudoviruses, Alpha pseudoviruses, Beta pseudoviruses, Gamma pseudoviruses, Delta pseudoviruses and Omicro pseudoviruses is 14.19 ng/mL, 14.12 ng/mL, 18.41 ng/mL, 15.59 ng/mL, 36.18 ng/mL and 19.26 ng/mL respectively. The ZW2G10 has broad-spectrum and high-efficiency neutralizing activity on the current main variant strain. The monoclonal antibody disclosed by the invention also has the characteristics of high expression, full human source and good stability, is suitable for industrial production, and has important application value for coping with outbreak and prevalence caused by new crown variants.

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 (COVID-19) is novel coronavirus-2 (SARS-CoV-2). RNA virus, its genome length is about 30 kb. 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 protein (E), membrane protein (M) and nucleocapsid protein (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 will enhance ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/565C07K2317/33C07K2317/76C07K2317/92A61K2039/505
Inventor 陈薇于长明李建民迟象阳孙韩聪张冠英张军范鹏飞张哲陈郑珊王步森宰晓东房婷付玲郝勐陈旖徐婧含
Owner ACADEMY OF MILITARY MEDICAL SCI
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