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Acquisition and application of anti-HCV (hepatitis C virus) antibody

A technology of antibodies and viruses, applied in the direction of antiviral agents, antiviral immunoglobulins, applications, etc., can solve problems such as unsuccessful marketing, and achieve the effect of reducing adverse reactions

Active Publication Date: 2017-10-24
GUANGZHOU TAINUODI BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The e20 antibody fragment was subsequently discovered to effectively neutralize infection of different HCV genotypes in vitro, but this research has not been successfully marketed until now

Method used

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  • Acquisition and application of anti-HCV (hepatitis C virus) antibody
  • Acquisition and application of anti-HCV (hepatitis C virus) antibody
  • Acquisition and application of anti-HCV (hepatitis C virus) antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0045] 1. Memory B cell sorting strategy

[0046] Flow Cytometry (FCM) is a high-speed, cell-by-cell quantitative analysis and sorting technique that represents a highly effective tool for the selection of high-affinity human monoclonal antibodies. In fact, flow-based sorting steps are very flexible and can be optimized to select for cross-reactive antibodies. In particular, HCV-I002 blood samples were obtained from confirmed HCV-infected patients, and peripheral blood mononuclear cells (Peripheral bloodmononuclear cells, PBMCs) were separated by lymphocyte separation tubes using a BD FACSria flow cytometer (BD Biosciences, San Jose, CA ) for cell sorting to obtain antigens specifically labeled with fluorescein to obtain E2 double fluorescently labeled target cells. Briefly, with this approach it is possible to obtain antibodies that are produced during natural infection but are still able to bind different glycoproteins that have never been encountered by the immune system o...

Embodiment 2

[0053] Example 2 Evaluation of neutralizing activity of TRN1007 antibody and different HCV true virus strains

[0054] Briefly, Huh7 cells were plated in 96-well plates and cultured overnight, and the cells were approximately 30% confluent at the time of infection. HCV true virus strains (h77, Con1, JFH1, S52, ED43, SA13, HK6a) were mixed with different concentrations of antibodies, the initial concentration was 25 μg / ml, diluted 3 times, and left at room temperature for 30 minutes. Add HCV true virus strains (h77, Con1, JFH1, S52, ED43, SA13, HK6a) and antibody mixture to a 96-well plate to infect Huh7 cells. In the control group, only HCV virus strain 2G9 was added, the medium was changed after 24 hours of infection, and the culture was continued for 1-2 days. The activity was measured 2-3 days after infection, and the fluorescence value was read. The neutralization efficiency was calculated by comparing the antibody with the control group.

[0055] The results are shown ...

Embodiment 3

[0058] Example 3 Assessment of Binding Activity of TRN1007 Antibody to HCV Antigens from Different Genotypes

[0059] Use different HCV strains (1a, 1b, 2, 3, 4, 5, 6, 7) membrane protein E2 as antigen, and use coating solution to dilute the antigen to a concentration of 100ng / ml, and coat it on an ELISA 96-well plate, 100 μl per well, overnight at 4°C. The blocking solution was blocked for 2 hours at 37°C. After blocking, add the primary antibody, the initial concentration is 25 μg / ml, 3-fold serial dilution, the volume of each well is 100 μl, and incubate at 37°C for 1 hour. At the same time, HCV positive patient serum is used as a positive control, and rabies antibody is used as a negative control. HRP-labeled goat anti-human IgG (diluted 1:2000) was used as a secondary antibody and incubated at 37°C for 1 hour. Add 100 μL / well of substrate chromogenic solution (TMB), place at 37°C in the dark for 5 min, stop the reaction with 2M sulfuric acid, and perform colorimetry wit...

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Abstract

The invention relates to acquisition and application of an anti-HCV (hepatitis C virus) antibody, in particular to fully-human monoclonal antibody TRN1007 or its functional fragment serving as a drug for treating and preventing HCV infections. The antibody TRN1007 acting as a spectral neutralizing antibody has a strong ability to bind with different antigens and a strong ability to neutralize different viruses, has high binding activity for viruses including genotypes 1a, 1b, 2, 3, 4, 5, 6 and 7, and has high neutralizing efficiency especially for HCV true strains S52 and HK6a, wherein IC50 value is only 4.6 nM and 6.6 nM. It is shown that the antibody TRN1007 with excellent binding and neutralizing activity has a promising application prospect in the detection, treatment and prevention of HCV infections and their related diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a fully human anti-HCV monoclonal neutralizing antibody and a preparation method and application of the neutralizing antibody. Background technique [0002] Hepatitis C virus (HCV) is still a major disease that threatens human health, affecting 2.0-3.0% of the world's population. HCV is a kind of yellow virus with a peripheral capsid (pericapsid) and single-stranded RNA. Viruses of the virus family. HCV is currently divided into 7 genotypes based on differences in their gene sequences, each marked with a number. Each genotype is further divided into several subtypes, each marked with a letter. Different HCV The prevalence and distribution of genotypes varies around the world. [0003] Hepatitis C virus is a hepatotropic virus that infects liver cells and causes liver lesions. Recently, some studies have shown that HCV infection can cause some pathological changes outside t...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13G01N33/569G01N33/577A61K39/395A61P1/16A61P31/14A61P35/00
CPCA61K2039/505C07K16/109C07K2317/21C07K2317/33C07K2317/56C07K2317/565C07K2317/76C07K2317/92G01N33/56983G01N33/577G01N2333/186G01N2469/10
Inventor 廖化新张远旭袁晓辉王月明昝利鹏吴昌文刘彤
Owner GUANGZHOU TAINUODI BIOTECH
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