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Antibody-peptide bispecific immunotherapeutic agent for Middle East respiratory syndrome coronaviruses

A bispecific antibody and antibody technology, applied in the direction of antiviral immunoglobulin, viral peptide, fusion polypeptide, etc., can solve the problems of limited application, high price, single target of monoclonal antibody, etc.

Inactive Publication Date: 2020-07-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, peptide drugs require chemical synthesis, and there are disadvantages such as unstable metabolism, short half-life in vivo, low bioavailability, and insufficient targeting, which seriously hinder the wide application of peptide drugs in clinical treatment.
On the other hand, the molecular weight of monoclonal antibody drugs is too large (150kDa), which leads to the disadvantages of poor tissue permeability, difficulty in reaching some sterically hindered targets, high production costs and expensive prices in clinical applications. Although monoclonal antibody drugs have achieved great success in the field of cancer and immune disease treatment, their application in antiviral treatment and other fields is relatively limited
In addition, monoclonal antibodies have a single target, which may cause virus escape

Method used

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  • Antibody-peptide bispecific immunotherapeutic agent for Middle East respiratory syndrome coronaviruses
  • Antibody-peptide bispecific immunotherapeutic agent for Middle East respiratory syndrome coronaviruses
  • Antibody-peptide bispecific immunotherapeutic agent for Middle East respiratory syndrome coronaviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation, expression and purification of antibody

[0040] Design two genes, one of which uses a linker (G4S)3 to connect the single-chain antibody (m336scFv) with an anti-viral polypeptide HR2P gene to construct a fusion protein m336scFv-pep, and the other uses a bivalent single-chain dimer diabody Linked with the polypeptide to construct the fusion protein m336diabody-pep, the gene was synthesized by the company and digested by SfiI, then inserted into the pComb3x expression vector by DNA ligase, and sent to the company for sequencing to verify the successful construction;

[0041] The preparation, expression, and purification methods of the fusion protein were carried out with reference to the method reported in the literature; the plasmids containing the clone m336scFv, the fusion protein m336scFv-pep, m336diabody-pep, and the fusion protein CH3-pep as a control were transferred into HB2151 competent cells, and from overnight Pick a single colony from t...

Embodiment 2

[0042] Example 2 Detection of Binding Ability of Bispecific Fusion Protein to S Protein of MERS Coronavirus

[0043] The S protein was fixed on the surface of the metal chip as a ligand, and four kinds of proteins m336scFv, m336scFv-pep, m336diabody-pep, and CH3-pep with a certain concentration gradient were passed through the chip at a certain flow rate to measure the change of refracted light. The reaction curve of the analyte and the substrate, and calculate the affinity constant (such as figure 2 shown), the fusion protein has a strong S protein binding ability.

Embodiment 3

[0044] Example 3 The bispecific fusion protein can effectively neutralize MERS-CoV

[0045] The MERS coronavirus S protein gene and the HIV-1 gene expressing luciferase as a reporter gene defect were co-transfected into 293T cells, and the MERS-CoV pseudovirus was packaged, and the proportionally diluted m336scFv, scFv-peptide and diabody-peptide and irrelevant antibody fusion protein CH 3 -peptide,, infect Huh-7 cells with pseudoviruses, measure luciferase activity, the results are as follows figure 1 As shown, IC of m336scFv, m336scFv-pep, m336diabody-pep 50 (μg / ml) were 0.011, 0.008 and 0.010, respectively, all of which could effectively neutralize MERS-CoV, among which m336scFv-pep had the highest neutralizing activity.

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Abstract

The invention belongs to the technical field of biology, and relates to fused protein of an antibody and an antivirus peptide, in particular to a bispecific efficient immunotherapeutic agent, of a fully human neutralizing antibody and a high-activity MERS-CoV-inhibiting peptide, for Middle East respiratory syndrome coronaviruses (MERS-CoV), and application of the bispecific efficient immunotherapeutic agent, of the fully human neutralizing antibody and the high-activity MERS-CoV-inhibiting peptide, for the Middle East respiratory syndrome coronaviruses (MERS-CoV). The antibody-peptide bispecific immunotherapeutic agent is the fused protein of the antibody and the antivirus peptide, wherein the antibody is the high-activity fully human neutralizing antibody to the MERS-CoV, and the peptideis the high-activity MERS-CoV-inhibiting peptide. The bispecific immunotherapeutic agent has antivirus activity of the antibody and the antivirus activity of the peptide at the same time, can be usedfor further producing a potent antivirus medicine, and is especially suitable for treating disease caused by the Middle East respiratory syndrome coronaviruses (MERS-CoV).

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fusion protein of an antibody and an antiviral polypeptide, in particular to a fully human neutralizing antibody for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and the bispecificity of a highly active MERS-CoV inhibitory polypeptide High-efficiency immunotherapeutic agent, and application thereof. Background technique [0002] Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a novel coronavirus discovered in June 2012 by virologist Ali M. Zaki [1]. Patients with MERS infection clinically show very similar symptoms to SARS, and most of them will develop severe respiratory syndrome, accompanied by a high rate of acute renal failure. The fatality rate is currently about 36%, which is much higher than during the SARS epidemic About 10% fatality rate [2,3]. Since 2012, MERS has spread to 26 countries. Among them, the outbreak in South Korea in 2015 developed very ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00G01N33/68G01N33/569A61K39/42A61K39/215A61P31/14
CPCC07K16/10C07K14/005G01N33/68G01N33/56983A61P31/14C07K2317/622C07K2317/92C07K2317/76C12N2770/20022C07K2319/00A61K39/00G01N2333/165G01N2469/10
Inventor 应天雷王丽丽吴艳玲
Owner FUDAN UNIV
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