Humanized monoclonal antibody of novel coronavirus and application thereof

A technology of cloning antibodies and source list, which is applied in the field of medicine, can solve the problems of no specific drugs being approved for marketing, and achieve the effect of good neutralizing activity

Active Publication Date: 2021-08-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Novel coronavirus 2019-nCoV is a new and sudden infectious disease pathogen, and there is currently no specific drug against the virus that has been approved for marketing

Method used

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  • Humanized monoclonal antibody of novel coronavirus and application thereof
  • Humanized monoclonal antibody of novel coronavirus and application thereof
  • Humanized monoclonal antibody of novel coronavirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Expression and purification of 2019-nCoV RBD

[0053] At the 3' end of the coding region of the 2019-nCoV RBD protein (the amino acid sequence is shown in SEQ ID NO: 9), connect the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon, and connect EcoRI and XhoI were constructed into pFastBacl vector (purchased from Invitrogen). The ligated product was then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. The recombinant baculovirus was extracted, transfected into sf9 cells (purchased from Invitrogen) for packaging of the baculovirus, and then amplified by the virus, added to Hi5 cells (purchased from Invitrogen) for 2019-nCoV RBD protein synthesis Express.

[0054] After the cell culture solution containing the target protein is purified by nickel ion affinity chromatography (HisTrap TMHP (GE)) and gel filtration chromatography (SuperoseTM 6 Increase10 / 300GL (GE)), a relatively pu...

Embodiment 2

[0055] Example 2: Isolation of 2019-nCoV RBD protein-specific memory B cells

[0056] With the informed consent of the discharged personnel after 2019-nCoV RBD infection, 15 mL of blood was collected to separate PBMCs. Separated PBMCs in 10 7 The density and final concentration of 400nM 2019-nCoV RBD protein was incubated on ice for half an hour, then washed twice with PBS, and then incubated with the following antibodies (both purchased from BD): anti-human CD3 / PE-Cy5, anti-human CD16 / PE-Cy5, anti-human CD235a / PE-Cy5, anti-human CD19 / APC-Cy7, anti-human CD27 / Pacific Blue, anti-human CD38 / APC, anti-human IgG / FITC, and anti-His / PE. After antibody incubation on ice for half an hour, PBMCs were washed twice with PBS.

[0057] After washing with PBS, PBMCs were sorted by FACSAria III to collect PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+ cells (i.e. B cells), and directly collected them into 96-well plates, 1 cell / well .

Embodiment 3

[0058] Example 3: Single B cell PCR, sequence analysis and human antibody design

[0059] According to the method described in Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus published in Science Translational Medicine, Volume 8, No. 369 in December 2016 by Qihui Wang et al., the B cells obtained in Example 2 were Reverse transcription was carried out by Superscript III reverse transcriptase (Invitrogen). The reverse transcription primers were listed in Table 1 and reacted at 55°C for 60 minutes.

[0060] Table 1. Primers for reverse transcription reactions

[0061]

[0062] Using this reverse transcription product as a template, PCR was performed with HotStar Tap Plus enzyme (QIAgen) to amplify the antibody variable region sequence (PCRa). Corresponding primers were designed, and the reaction conditions were as follows: 95°C, 5min; 95°C for 30s, 55°C (heavy chain / κ chain) for 30s, 72°C for 90s, 35 cycles; 72°C, 7min...

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Abstract

The invention relates to a novel human monoclonal antibody of coronavirus and application of the novel human monoclonal antibody. The antibody can be specifically combined with the 2019-nCoV RBD, the combination of the 2019-nCoV RBD and ACE2 is blocked, and the infection of the 2019-nCoV is inhibited.

Description

technical field [0001] The invention belongs to the field of medical technology, and in particular relates to a novel coronavirus (2019-nCoV) human monoclonal antibody with high neutralization activity and its application. Background technique [0002] The novel coronavirus 2019-nCoV is a new and sudden infectious disease pathogen, and there is currently no specific drug against the virus that has been approved for marketing. [0003] Therapeutic antibody drugs not only play an important role in tumors and autoimmune diseases, but are also effective in the treatment of infectious diseases. Drugs currently on the market for the treatment and prevention of viral infections include palivizumab (Synagis) for the prevention of pediatric respiratory syncytial virus (RSV) infection, ibalizumab (Trogarzo) for the treatment of HIV infection, and rabies Rabishield for post-exposure prophylaxis. There are also monoclonal antibodies against numerous viruses in various stages of clinic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/56C07K2317/92C07K2317/76A61K2039/505
Inventor 王奇慧严景华史瑞高福马素芳
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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