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Humanized broad-spectrum high-neutralizing-activity monoclonal antibody against novel coronavirus and application

A technology of monoclonal antibody and coronavirus, applied in the field of peptides, to achieve significant broad-spectrum neutralization ability and good stability

Pending Publication Date: 2021-10-19
THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is necessary to isolate more potent neutralizing antibodies as alternatives, to combine these neutralizing antibodies against different epitopes, and to explore cocktail therapy, which can more effectively prevent the virus from escaping. No similar broad-spectrum antibody and antibody composition have been reported yet

Method used

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  • Humanized broad-spectrum high-neutralizing-activity monoclonal antibody against novel coronavirus and application
  • Humanized broad-spectrum high-neutralizing-activity monoclonal antibody against novel coronavirus and application
  • Humanized broad-spectrum high-neutralizing-activity monoclonal antibody against novel coronavirus and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Synthesis, expression, biotinylation and staining of the novel coronavirus RBD probe

[0036] 1.1 According to the data published by Genbank (NC_045512), the synthesis carried 6×His-Avi (His-His-His-His-His-His-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu- As p-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-Asp-Ile-Thr-Asn-Trp-Leu-Trp-Tyr-I Le-Lys-Lys-Lys) tagged RBD full long gene sequence.

[0037] 1.2 Reconnected into the eukaryotic expression vector pDRVI1.0 (constructed and preserved by the inventor) after EcoRI and EcoRV double digestion, and the sequence was correct after the clone was selected.

[0038] 1.3 The two probe plasmids were respectively transfected into 293F cells for expression. After 5-6 days, the culture medium was centrifuged to collect the cell supernatant, and the antigenic protein was purified through a nickel column.

[0039] 1.4 Use BirA 500 Biotin Protein Ligase Kit (BirA500, Avidity) to biotinylate the probe protein.

[0040] Dissolve 1 mg mo...

Embodiment 2

[0046] Embodiment 2: screening and identification of anti-SARS-COV-2 humanized monoclonal antibody

[0047] 2.1 Prepare cell lysate: 20 μl cell lysate per well, including 0.5 μl RNase removal, 5 μl 5×FirstStrand buffer, 1.25 μL 0.1M DTT, 0.0625 μl Igepal, 13.25 μl water, cover the plate with sealing film, and refrigerate at 4°C Set aside.

[0048] 2.2 Sample preparation:

[0049] (1) Resuscitation of PBMCs cells from recovered patients after COVID-19 infection: After taking out the frozen cell tube from liquid nitrogen, quickly place it in a 37°C water bath, take it out when it melts until there is an ice core, open it in a biological safety cabinet, and slowly Add R10+ Benzonase medium dropwise (5mL R10+ Benzonase medium is used for 1 branch of cells). Centrifuge at 1500rpm for 10 minutes, discard the supernatant, suspend the cells with the residual solution, add 10mL R10, mix well, take 50μl for cell counting, and centrifuge at 1500rpm for 10 minutes; adjust the cell conce...

Embodiment 3

[0102] Embodiment 3 antibody is to the neutralizing activity determination of SARS-COV-2 pseudovirus

[0103] 3.1 Packaging of pseudovirus: The full-length S protein gene of artificially synthesized SARS-CoV-2 (GenBank: MN908947) was inserted into the pcDNA3.1 expression plasmid to construct pcDNA-SARS-CoV2-S. The pseudovirus mutation site with mutation is introduced on the S gene as shown in table 11.

[0104] Table 11. Pseudovirus mutation sites

[0105]

[0106]

[0107] will be 3×10 6 (3 million) 293T / 17 cells were inoculated in T75 cell culture flasks, 5% CO 2 Incubate at 37°C for 20-24 hours. Use Fugene 6Transfection Reagent (Promega, Cat#E2691) for transfection: 30ug plasmid pcDNA-SARS-CoV2-S was transfected into 293T cells in a T75 culture flask, and 1.05×10 6 The G*ΔG-VSV virus of TCID50 infected 293T, and the medium was changed after 8 hours. After 24 hours of transfection, the culture supernatant was collected and filtered to obtain the pseudovirus of th...

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Abstract

The invention discloses a group of humanized broad-spectrum monoclonal neutralizing antibodies for resisting SARS-COV-2 virus, and the neutralizing antibodies are obtained by screening through a single B cell flow sorting-antibody gene amplification pairing expression technology and have a unique CDR region; theneutralizing antibodies can be specifically combined with SARS-COV-2 and can effectively neutralize a plurality of international epidemic virus strains (a novel coronavirus mutant strain A, a novel coronavirus mutant strain B.1. 1.7, a novel coronavirus mutant strain B.1.351, a novel coronavirus mutant strain P.1, a novel coronavirus mutant strain B.1.617.1 and a novel coronavirus mutant strain B.1.617.2) at present, wherein the IC50 is about 0.1 [mu]g / mL. The present invention also relates to methods of preparation and uses of the set of neutralizing antibodies. The three antibodies have the effect of synergistically neutralizing viruses when being used in a pairwise combined manner, so that the combination of the three antibodies can be used for emergency prevention and / or treatment of COVID-19, has the characteristics of full humanization, high expression and good stability, and is suitable for industrialization. In addition, the antibody can also be used for preparing an SARS-COV-2 virus detection reagent, finding effective neutralizing epitopes and developing SARS-COV-2 recombinant protein and subunit vaccines.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an antibody. Background technique [0002] SARS Cov-2 (also known as 2019-nCov) is a positive-strand RNA virus belonging to the β genus of the coronavirus family, which encodes four structural proteins: spike (S), envelope (E), membrane (M) , and nucleocapsid (N), 16 non-structural proteins, and 5-8 auxiliary proteins. SARS Cov-2 uses the S protein on the surface of the virus and the host cell receptor-angiotensin-converting enzyme II (ACE2) to enter cells. S protein is divided into two functional units according to protein structure and function, namely S1 and S2 protein subunits. S1 can be divided into NTD (N-terminal domain) and RBD (Receptor binding site). The RBD region is about 240 amino acids long and mainly binds to host cell receptors. S2 plays a role in the fusion of virus and cell membranes. According to existing reports, the neutralizing antibody mainly act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/85C12N15/13C12N5/10G01N33/569G01N33/577A61K39/42A61P31/14
CPCC07K16/10C12N15/85C12N5/0686G01N33/56983G01N33/577A61P31/14C07K2317/565C07K2317/24C12N2510/02G01N2333/165G01N2469/10C07K2317/56C07K2317/52C07K2317/92C12N2800/107A61K2039/505
Inventor 邵一鸣朱彪李丹吴南屏王铮郝彦玲
Owner THE FIRST AFFILIATED HOSPITAL ZHEJIANG UNIV COLLEGE OF MEDICINE
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