Single-domain antibodies for novel coronavirus and application of single-domain antibodies

A single-domain antibody and coronavirus technology, applied in the field of biomedicine, can solve the problems of low homology and lack of cross-protection effect, and achieve the effect of good affinity and high neutralization activity

Active Publication Date: 2020-06-19
BEIJING KAWIN TECH SHARE HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After gene sequence comparison analysis, the homology of the RBD region of SARS-CoV-2 with the common cold coronavirus and MERS-CoV is extremely low, and there are a large number of mutations even compared with the RBD region of SARS-CoV, so the known targets for General lack of cross-protection between neutralizing antibodies to SARS-CoV and MERS-CoV and the emerging pathogen of SARS-CoV-2

Method used

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  • Single-domain antibodies for novel coronavirus and application of single-domain antibodies
  • Single-domain antibodies for novel coronavirus and application of single-domain antibodies
  • Single-domain antibodies for novel coronavirus and application of single-domain antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1. Screening and preparation of single-domain antibodies that recognize the RBD region of the SARS-CoV-2 spike protein

[0071] 1. Phage library screening

[0072] The basic principle and basic operation process of phage library screening refer to "Biological Library Technology-Phage Display and SELEX Technology" written by Shao Ningsheng et al., and the brief description is as follows: First, 50 μg of recombinant protein in the RBD region of the SARS-CoV-2 spike protein ( Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat. No.: 40592-V08H; hereinafter referred to as RBD protein) was biotin-labeled with EZ-LinkTMSulfo NHS-LC-LC-Biotin, a commercial kit from Thermo Fisher Scientific, and desalted and purified (The desalting column was purchased from Thermo Fisher Scientific). The first round of screening was liquid-phase selection, firstly, 12 μg of biotin-labeled RBD protein was mixed with 1 mL (10 12 -10 13 ) phage library and incubate at room temperature for...

Embodiment 2

[0104] Example 2, Affinity Determination of Anti-RBD Single Domain Antibody

[0105] Antibodies to be tested: five single domain antibodies prepared in Example 1.

[0106] BIAcore T-200 biomolecular interaction instrument (GE Life Sciences) was used to measure the affinity of antibodies. This technology was developed based on surface plasmon resonance SPR technology, which can perform label-free, fast and real-time interaction between biomacromolecules. , Automatic operation and detection.

[0107] When measuring the interaction between RBD protein and its corresponding single-domain antibody, the RBD protein was first coated on the sensor chip, and then the single-domain antibody was used as the mobile phase to measure the association constant, dissociation constant and affinity constant.

[0108] 1. RBD protein coupling CM5 chip: RBD protein coupling temperature is 25 degrees Celsius, the buffer is HBS-P (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20, pH 7.4). For the pro...

Embodiment 3

[0114] Example 3, SARS-CoV-2 pseudotyped virus neutralizing activity assay of anti-RBD single domain antibody

[0115] Antibodies to be tested: five single domain antibodies prepared in Example 1.

[0116]SARS-CoV-2 pseudotyped virus is a new type of virus particle formed by assembling the replication core element of retrovirus and the envelope spike glycoprotein (ie, S protein) of SARS-CoV-2 virus. The ability of pseudotyped virus to infect cells depends on the type and characteristics of the glycoprotein wrapped outside it, and it is an ideal tool for studying the inhibition efficiency of neutralizing antibodies, receptor utilization and invasion infection mechanism of SARS-CoV-2.

[0117] 1. Packaging preparation of SARS-CoV-2 pseudotyped virus

[0118] 293T cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated in a 10 cm culture dish and cultured in DMEM medium (purchased from Thermo Fisher) containing 10% fetal bovine s...

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Abstract

The invention discloses humanized single-domain antibodies for a novel coronavirus SARS-CoV-2 and an application of the humanized single-domain antibodies. The invention protects the single-domain antibodies described in any one of SEQ ID No.1 to SEQ ID No.5. Experimental results show that the single-domain antibodies provided by the invention have good affinity with receptor binding domain (RBD)antigens and have high neutralization activity on SARS-CoV-2 pseudovirus. The humanized single-domain antibodies have important scientific significance and application prospect for prevention and clinical treatment of the new coronavirus SARS-CoV-2 and for development of diagnostic reagents of the new coronavirus SARS-CoV-2.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a humanized single-domain antibody against novel coronavirus SARS-CoV-2 and its application. Background technique [0002] Antibodies are a class of immunoglobulins that can specifically bind to antigens. Antibody drugs generally refer to the full antibody molecules and antibody fragments with therapeutic functions obtained by advanced technology, and are one of the important means of targeted therapy. The specific targets of antibody drugs include but are not limited to: cancer cell specific antigens, immune checkpoint molecules, cytokines and their receptors, viral proteins, bacterial toxins, etc. The main types of diseases treated by antibody drugs include various cancers, blood diseases, autoimmune diseases such as rheumatoid arthritis, cardiovascular diseases, and viral infections. In addition to being used as a therapeutic method, antibodies are also often used in the fields of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/569C07K2317/565C07K2317/567C07K2317/92C07K2317/76A61K2039/505C12N15/70G01N33/56983C12N2800/101G01N2333/165G01N2469/10Y02A50/30
Inventor 杨威迟晓静刘秀英
Owner BEIJING KAWIN TECH SHARE HLDG
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