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Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody

A coronavirus and antibody technology, applied in the field of immunity, can solve problems such as drug resistance and reduced neutralization activity, and achieve the effect of avoiding biosafety risks

Active Publication Date: 2022-05-31
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the anti-SARS-CoV-2 neutralizing antibodies that are currently approved or in different research stages have no significant reduction or only partially reduce the neutralizing activity against mutant strains such as Alpha, Beta, Gamma and Delta, but more than 85% of the antibodies have Omicron mutants are almost completely drug-resistant

Method used

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  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody
  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody
  • Broad-spectrum neutralizing antibody for resisting novel coronavirus and application of broad-spectrum neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Preparation of anti-novel coronavirus monoclonal antibodies

[0079] 1. Detection of plasma binding activity

[0080] 1. Sample: plasma of volunteers immunized twice with novel coronavirus inactivated vaccine (BBIBP-CorV).

[0081] 2. Detection of binding activity: The binding titers of 28 samples to the RBD protein of the novel coronavirus Omicron mutant strain (B.1.1.529) were detected. Methods as below:

[0082] (1) Coating: Dilute the above RBD protein with PBS to 0.5 µg / ml, add 100 µl / well to a 96-well ELISA plate, and coat overnight at 4°C.

[0083] (2) Blocking: discard the coating solution, add 200 µl blocking solution (PBST+3%BSA) to each well, and block at 37°C for 1 h.

[0084] (3) Washing: The blocking solution was discarded and washed three times with PBST.

[0085] (4) Sample: each plasma sample was diluted 100 times, serially diluted 3 times, and a negative control was added, and incubated at 37°C for 2 h.

[0086] (5) Washing: The sample ...

Embodiment 2

[0173] Example 2. Preparation of monoclonal antibodies

[0174] 1. Antibody expression in different cell lines

[0175] 1. Antibody expression in 293T cell system

[0176] Transient transfection of successfully paired antibody heavy and light chain gene expression vectors into 293T cells:

[0177] (1) 24 hours before transfection in a 12-well plate, each well was plated with 5 × 10 5 293T cells.

[0178] (2) Observe the degree of confluence of cells on the day of transfection, preferably 70% to 80%.

[0179] (3) Take 1 μg of each plasmid DNA extracted by the plasmid mini-extraction kit (Tiangen, DP103) and dilute it with 60 μl of Opti-MEM medium.

[0180] (4) Dilute 4 μl Lipofectamine® 2000 Reagent with 60 μl Opti-MEM medium.

[0181] (5) Mix the above-diluted DNA and Lipofectamine® 2000 at a volume of 1:1 to obtain a mixed solution, and incubate at room temperature for 5 min.

[0182] (6) Gently add 120 μl of the mixture to 293T cells, 5% CO 2 Incubate at 37°C.

[01...

Embodiment 3

[0242] Example 3. Antibody 10-5B Affinity and Neutralizing Activity Analysis

[0243] 1. Affinity analysis of monoclonal antibodies

[0244] 1. BLI detects the binding ability of monoclonal antibody 10-5B to 2019-nCoV RBD

[0245] (1) Ligand coupling: Biotin-labeled 10 µg / ml wild-type RBD protein (acrobiosystems, SPD-C82E9) and Omicron (Omicron) were mutated on Octet RED 384, a high-throughput intermolecular interaction instrument. The RBD protein of the strain (Sino Biological, 40592-V49H7-B) was coupled to the SA sensor (SARTORIUS, 18-0009).

[0246] (2) Antibody 10-5B was serially diluted 2-fold in PBS from 160 nM in 6 gradients.

[0247] (3) Set the injection analysis from low concentration to high concentration according to Table 5 below.

[0248] Table 5. Low to High Injection Settings

[0249]

[0250] (4) Data analysis: The following Table 6 settings are performed in the analysis software, and curve fitting is performed.

[0251] Table 6 Analysis software setti...

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Abstract

The invention relates to a broad-spectrum neutralizing antibody for resisting novel coronavirus and application of the broad-spectrum neutralizing antibody. The invention provides an anti-novel coronavirus broad-spectrum neutralizing antibody or an antigen binding fragment thereof, the broad-spectrum neutralizing antibody or the antigen binding fragment thereof has a heavy chain variable region containing VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region containing VLCDR1, VLCDR2 and VLCDR3, the VHCDR1, the VHCDR2 and the VHCDR3 respectively comprise amino acid sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, and the VLCDR1, the VLCDR2 and the VLCDR3 respectively comprise amino acid sequences shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6. The finally screened antibody has efficient and broad-spectrum neutralizing activity, the IC50 of the antibody to a wild type of the novel coronavirus and 13 mutant strains is less than 0.05 g / ml, and the IC50 of the antibody to WT, Beta, Delta and Omicro BA.1 live viruses is less than 0.1 g / ml.

Description

technical field [0001] The invention relates to the technical field of immunity, in particular to broad-spectrum neutralizing antibodies against novel coronaviruses and applications thereof. Background technique [0002] The novel coronavirus (SARS-CoV-2) randomly mutates to form various mutant strains during the transmission process, and some of the mutant strains carrying key mutation sites have stronger immune evasion ability, thus causing breakthrough infection. Take the Omicron mutant strain (B.1.1.529) as an example, a large number of mutations in important target sites greatly reduce the neutralizing activity of immune serum, posing a threat to the protective effect of the vaccine. Therefore, efficient and broad-spectrum preventive and therapeutic approaches are urgently needed to control the SARS-CoV-2 epidemic. Broadly neutralizing antibodies (bNAbs) can effectively deal with the immune escape of different mutant strains, which can not only be administered after in...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/56C07K2317/565C07K2317/52C07K2317/76C07K2317/92A61K2039/505
Inventor 程功刘玉斌邹艳盛洁
Owner TSINGHUA UNIV
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