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High-neutralizing-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody and application thereof

A monoclonal antibody, fully human technology, applied in the field of microbiology and immunology, can solve the problems of difficult large-scale preparation, batch differences, limited plasma donation sources, etc., achieve low immunogenicity, good stability, good The effect of neutralizing the protective effect

Active Publication Date: 2020-06-19
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages: limited sources of donated plasma, difficulty in large-scale preparation, batch-to-batch differences, etc.; whether there is antibody-dependent enhancement (ADE effect) is still controversial

Method used

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  • High-neutralizing-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody and application thereof
  • High-neutralizing-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody and application thereof
  • High-neutralizing-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening and Preparation of Human Anti-SARS-CoV-2 Monoclonal Antibody

[0032] 1.1 Sorting single cells by flow cytometry

[0033] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:

[0034] 1.1.1 Take fresh anticoagulated whole blood (EDTA anticoagulated) and dilute the whole blood with an equal volume of PBS.

[0035] 1.1.2 Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1.

[0036] 1.1.3 Trim, room temperature, horizontal rotor 700-800g (2000-2500rpm), acceleration 3-4 acc, centrifuge for 20-30min.

[0037] 1.1.4 After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation l...

Embodiment 2

[0091] Example 2 Screening of binding activity of human anti-SARS-CoV-2 monoclonal antibody to S protein and analysis of antibody 4A8 recognition epitope

[0092] 2.1 Coating: Dilute the recombinant S antigen, S1 antigen, RBD antigen and S2 antigen with the coating solution to a concentration of 2 μg / mL, coat the microtiter plate, 100 μL per well, and coat overnight at 4 °C.

[0093] 2.2 Blocking: add 300 μL of PBST washing solution to each well, wash 3 times × 3 min each time; tap the liquid in the well, add 2% BSA, 200 μL / well, block at 37°C for 1 hour.

[0094] 2.3 Sample incubation: Add 300 μL PBST washing solution to each well, wash 3 times × 3min / time; pat the liquid in the well, add purified monoclonal antibody diluted in PBS, the first well 9ug / ml, 3-fold serial dilution 100 μL / well, 37 Incubate at ℃ for 1h.

[0095] 2.4 Secondary antibody incubation: wash, same as above; add HRP goat anti-human F C Secondary antibody (diluted 1:20000), 100 μL / well, incubated at 37°C...

Embodiment 3

[0102] Neutralizing activity analysis on cell model during embodiment 3 SARS-CoV-2 infection

[0103] 3.1 After digesting Vero E6 cells with 0.25% trypsin, dilute to 3×10 with medium (DMEM+10% FBS) 5 The concentration of cells / mL was inoculated into 96-well cell culture plates with an inoculation volume of 100 μL / well, and cultured overnight in a 5% CO2 cell incubator at 37°C.

[0104] 3.2 On the day of the experiment, add the purified monoclonal antibody to the 96-well culture plate from the initial concentration (4A8 monoclonal antibody initial concentration 154ug / ml, 3-fold serial dilution, volume 120μL / well; then add 120μL per well COVID-19 virus suspension (dilute virus with DMEM+2%FBS, add 100TCLD 50 / well), mix thoroughly, and incubate for 1 h in a cell culture incubator.

[0105] 3.3 Discard the cell culture supernatant in the 96-well plate, and add 200 μL of co-incubated virus-antibody mixed suspension to each well; set up a survival control (no virus and antibody) ...

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Abstract

The invention discloses an anti-SARS-CoV-2 fully-humanized monoclonal antibody against SARS-CoV-2. The antibody is obtained by screening through a flow sorting-single cell PCR technology, and has a unique CDR partition. The invention further discloses an application of the antibody in preparation of medicines for treating the 2019-coronavirus disease. The monoclonal antibody disclosed by the invention has efficient and specific anti-SARS-COV-2 activity, also has the characteristics of high expression, complete humanization and good stability, and is suitable for industrial production.

Description

technical field [0001] The invention discloses an antibody and belongs to the fields of microbiology and immunology. Background technique [0002] The causative agent of COVID-19 is SARS-CoV-2, also known as 2019-coronavirus. SARS-CoV-2 belongs to the Coronavirus family (Coro-naviridae) and is a class of single-stranded positive-sense RNA viruses with an envelope. The total length of the genome is 30kb, which consists of 5'-terminal non-coding region, non-structural protein open reading frame (ORF) 1a / b coding region, spike glycoprotein S coding region, envelope protein E coding region, membrane protein M protein Coding region, nucleocapsid protein N protein coding region and 3'-terminal non-coding region. Among them, the polyprotein encoded by the non-structural protein ORF1a / b region can be cleaved to form RNA-dependent RNA polymerase (RdRp), etc., to guide viral genome replication, transcription and translation. Structural protein S can specifically bind to receptors o...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/52C07K2317/92C07K2317/34C07K2317/565C07K2317/76A61K2039/505
Inventor 陈薇李建民迟象阳张军付玲于长明徐俊杰侯利华张冠英范鹏飞郝勐董韵竹宋小红陈旖张金龙房婷刘树玲
Owner ACADEMY OF MILITARY MEDICAL SCI
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