Neutralizing antibody against novel coronavirus receptor binding regions and application thereof

A coronavirus, receptor binding technology, applied in the direction of antiviral agents, viruses/phages, antiviral immunoglobulins, etc., to achieve the effect of improving the neutralization ability

Active Publication Date: 2021-07-23
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

X.Sunney Xie, Chen Wei, Linqi Zhang et al obtained some high-efficiency neutralizing antibodies by using single-cell sequencing and single-cell PCR technology after enriching B cells by antigen, but they have not been used in clinical practice, and there is no therapeutic effect yet Reports that antibodies have been applied to disease treatment practice through clinical approval, so the research and development of protective neutralizing antibodies still needs to be carried out, which can not only provide candidate targets for the application of therapeutic antibodies, but also provide ideas for vaccine design

Method used

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  • Neutralizing antibody against novel coronavirus receptor binding regions and application thereof
  • Neutralizing antibody against novel coronavirus receptor binding regions and application thereof
  • Neutralizing antibody against novel coronavirus receptor binding regions and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 110

[0105] Example 1 10×Genomics database construction and data analysis

[0106] In this example, the peripheral blood mononuclear cells of patients with COVID-19 were first extracted by lymphocyte separation medium, and CD19 阳性 CD27 阳性 Memory B cells and CD19 阳性 CD27 高 CD38 高 Plasma cells, and label B cells specific to the receptor binding region of the new coronavirus;

[0107] The prepared cells were subjected to 10× Genetics library construction and sequencing, and three libraries were constructed: 5' transcriptome library, B cell receptor library and signature library, and finally high-throughput sequencing was performed using NovaSeq 6000;

[0108] Use 10×Genomics Cell Ranger software (version 3.1.0) to process sequencing fastq files, use Seurat (version 3.1) to filter low-quality cells (gene expression 5000, mitochondrial genes>10%, gene expression less than 3 cells), and finally use Seurat for standardization, PCA dimensionality reduction, TSNE clustering, and differ...

Embodiment 2

[0109] Example 2 Synthesis and expression of antibody

[0110] Candidate antibodies were selected according to the following principles: 1) antibody clonal types other than IgE and IgD, 2) clones with high and independent antigen labeling, 3) clones with high somatic mutation rate excluded, 4) clones with high-frequency V-J combinations. Antibody C14646P3S (SEQ ID NOs: 7-8) and antibody C2767P3S (SEQ ID NOs: 17-18) were obtained from this screening.

[0111] The heavy chain CDR3 of C14646P3S was further mutated to obtain neutralizing antibody mutants C14646P3S_M1 (SEQ ID NO:22, SEQ ID NO:8), C14646P3S_M2 (SEQ ID NO:25, SEQ ID NO:8), C14646P3S_M3 (SEQ ID NO: 28, SEQ ID NO: 8).

[0112] The screened antibody gene was linked to an IgG carrier containing a constant region, and the antibody was expressed by transfecting HEK293T cells, adding a transfection reagent, collecting the supernatant and performing antibody purification.

Embodiment 3

[0113] Specific identification of embodiment 3 antibody

[0114] This embodiment utilizes ELISA to identify the specificity of the antibody, prepares a microtiter plate coated with the SARS-CoV-2 receptor binding region antigen, and adds screening antibody C14646P3S and its mutant antibody C14646P3S_M1, C14646P3S_M2, C14646P3S_M3, C2767P3S or positive control antibody (S309 ), followed by the addition of HRP-labeled goat anti-human IgG secondary antibody for ELISA detection.

[0115] The result is as figure 1As shown, C14646P3S and its mutant antibodies C14646P3S_M1, C14646P3S_M2, C14646P3S_M3 and C2767P3S monoclonal antibodies strongly bind to the receptor binding region.

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Abstract

The invention provides a neutralizing antibody against novel coronavirus receptor binding regions and application thereof, and belongs to the technical field of biological products. The neutralizing antibody comprises a heavy chain variable region and a light chain variable region; and the heavy chain variable region comprises a heavy chain CDR3 as shown in SEQ ID NO: 3 or SEQ ID NO: 13, and the light chain variable region comprises a light chain CDR3 as shown in SEQ ID NO: 6 or SEQ ID NO: 16. According to the neutralizing antibody against the novel coronavirus receptor binding regions and the application thereof, the neutralizing antibodies C14646P3S and C2767P3S of the SARS-CoV-2 receptor binding region are obtained by utilizing LIBRA-seq high-throughput screening; and the neutralizing antibodies C14646P3S and C2767P3S have relatively strong neutralizing capability on SARS-CoV-2 pseudoviruses and live viruses in vitro, can well block the binding of a novel coronavirus receptor binding region and a receptor thereof, and have important application value in the aspects of prevention, treatment and diagnosis of SARS-CoV-2 infection.

Description

technical field [0001] The invention belongs to the technical field of biological products, and relates to an anti-new coronavirus receptor binding domain neutralizing antibody and its application. Background technique [0002] The novel coronavirus SARS-CoV-2 is an enveloped single positive-strand RNA virus belonging to the coronavirus β-CoV family. The genome mainly encodes four structural proteins, about 16 non-structural proteins (nap1-16) and 5-8 There are four kinds of auxiliary proteins, among which the four structural proteins are spike protein (S protein), envelope protein (E protein), membrane protein (M protein) and nucleocapsid protein (N protein). Among the four structural proteins, the S protein plays a crucial role in the adsorption, membrane fusion, invasion and spread of SARS-CoV-2 to host cells. The S protein includes the S1 subunit involved in virus-receptor binding and the S2 subunit involved in viral membrane fusion, and the S1 subunit is further divide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14A61P11/00C12N15/85C12N5/10G01N33/569G01N33/68
CPCC07K16/10A61P31/14A61P11/00C12N15/85G01N33/56983G01N33/6854C07K2317/565C07K2317/56C07K2317/76C07K2317/14C12N2800/107A61K2039/505G01N2469/10G01N2333/165
Inventor 陈耀庆何兵刘舒宁王媛媛
Owner SUN YAT SEN UNIV
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