Fully-humanized monoclonal antibody against chikungunya fever and with high neutralizing activity, and application thereof

A monoclonal antibody, chikungunya virus technology, applied in the field of microbiology and immunology, can solve problems such as non-specific therapeutic drugs, achieve low immunogenicity, improve efficacy, and avoid the generation of drug-resistant strains Effect

Active Publication Date: 2020-03-24
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the treatment of chikungunya fever is mainly symptomatic treatment such as fever reduction and analgesia, and there is no specific therapeutic drug in clinical practice.

Method used

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  • Fully-humanized monoclonal antibody against chikungunya fever and with high neutralizing activity, and application thereof
  • Fully-humanized monoclonal antibody against chikungunya fever and with high neutralizing activity, and application thereof
  • Fully-humanized monoclonal antibody against chikungunya fever and with high neutralizing activity, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Screening and Preparation of Human Anti-Chikungunya Virus Monoclonal Antibody

[0030] 1.1 Blood sample collection

[0031] After obtaining informed consent, the Guangzhou Eighth People's Hospital provided the peripheral blood of a chikungunya infected patient 15 days after recovery for subsequent experiments.

[0032] 1.2 Sorting single cells by flow cytometry

[0033] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation. The process is as follows:

[0034] 1.2.1 Take fresh anticoagulated whole blood (EDTA anticoagulation) and dilute the whole blood with an equal volume of PBS.

[0035] 1.2.2 Add a certain volume of separation liquid to the centrifuge tube, and spread the diluted blood sample above the liquid surface of the separation liquid to keep the interface between the two liquid surfaces clear. Separation solution, anticoagulated undiluted whole blood, PBS (or normal saline) volume is 1:1:1.

[0036] 1.2.3...

Embodiment 2

[0118] Example 2. Cell Neutralization Experiment

[0119] 2.1 The day before the experiment, the Vero cells were diluted to 1.5×10 with medium (MEM+10%FBS) 5 cells / mL concentration, inoculated into 96-well cell culture plate, inoculated volume 200μL / well, placed in 37°C 5% CO2 cell incubator;

[0120] 2.2 On the day of the experiment, the purified monoclonal antibody was used in the medium MEM+2% FBS from the initial concentration (the initial concentration of 8D1 monoclonal antibody was 100ug / ml, and the Ab2 control antibody was 200ug / ml (refer to the variable heavy chain and light chain of 8B10F8 disclosed in U.S. Patent US9738704). Region sequence, chemically synthesized variable region gene and obtained full-length antibody gene by fusion PCR technology, cloned into the expression vector pMH vector, transfected into CHO-S cells, purified by Protein A affinity chromatography, obtained and stored, named after the laboratory Ab2), diluted 4 times, added to a 96-well culture ...

Embodiment 3

[0125] Example 3. Monoclonal antibody recognition epitope research

[0126] 3.1 ELISA to screen specific clones: Coat 96-well enzyme-linked plate with anti-chikungunya virus monoclonal antibody (100 μg / mL, 150 μL / well), overnight at 4°C; discard the coating liquid and add 5 mg / mL BSA to block The buffer solution (0.1mol / L NaHCO3, pH8.6) was blocked at 4°C for 2h; the liquid was poured off, washed 6 times with TBST (1mL / L Tween-20, TBS); 100 μL of the phage loop 7 peptide library was added (the original library used TBST Diluted at 1:10, containing about 2 × 10 bacteriophages 11 pfu / 100 μL), incubate with gentle shaking at room temperature for 30 min; discard the liquid, wash 10 times with TBST, and wash away unbound phage; add 100 μL eluate (1 mg / mL BSA, 0.2 mol / L Glycine-HCl, pH 2.2 ) elution, shake gently for 10 min at room temperature, quickly aspirate the liquid and add 15 μL of neutralizing solution (1 mol / L Tris-HCl, pH 9.1) to neutralize; take 1 μL of the eluted phage ...

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Abstract

The invention discloses a fully-humanized monoclonal antibody against the chikungunya virus and with high neutralizing activity. The antibody is obtained by screening via a flow sorting-single cell PCR technology and has unique CDR subregions. The invention also discloses the application of the antibody in preparation of treatment drugs for chikungunya fever. The monoclonal antibody disclosed by the invention has efficient and specific anti-chikungunya virus activity, also has the characteristics of high expression, full human origin and good stability, and is suitable for industrial production.

Description

technical field [0001] The invention discloses an antibody, which belongs to the fields of microbiology and immunology. Background technique [0002] Chikungunya fever (chikungunya fever) is a mosquito-borne infectious disease caused by chikungunya virus, initially prevalent in tropical and subtropical regions of Africa, and continues to expand to South Asia, Southeast Asia, Indian Ocean islands and Americas region. The main clinical signs of chikungunya are fever, headache, muscle pain, rash and joint pain. In the past ten years, the number of outbreaks of Chikungunya fever has increased and the epidemic scope has continued to expand. It has occurred in more than 100 countries and regions around the world, causing about 1 million cases worldwide each year. On December 20, 2015, the World Health Organization identified eight dangerous pathogens (Ebola, Marburg virus, etc.) and three less dangerous pathogens (Chikon) that could lead to severe outbreaks based on lessons lear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13A61P31/14
CPCA61P31/14C07K16/1081C07K2317/24C07K2317/52C07K2317/56C07K2317/565C07K2317/76Y02A50/30
Inventor 陈薇李建民迟象阳付玲于长明徐俊杰侯利华宋小红刘树玲董韵竹张金龙房婷张复春
Owner ACADEMY OF MILITARY MEDICAL SCI
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