Specific HBV antibody

A specificity and antibody technology, applied in the direction of antibodies, specific peptides, antiviral agents, etc., can solve the problems of low specific activity, unknown antigen, insufficient supply, etc., achieve high affinity, reduce viral load, and low allergic reaction original effect

Inactive Publication Date: 2012-11-21
傅阳心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although both HBIg and HBV monoclonal antibodies can effectively neutralize HBV, there are many problems with the use of HBIg: such as insufficient supply, low specific activity, potential contamination of unknown antigens, etc.
While fully human m

Method used

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  • Specific HBV antibody
  • Specific HBV antibody
  • Specific HBV antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1 Screening of specific scFv against adr and adw subtype HBsAg

[0112] I. Screening of high-affinity specific scFv

[0113] Using a non-immune human scFv library (see Nature Protocols Vol. 1 No. 2: 755-768, 2006 for specific information about this library), the scFv against adr and adw subtype HBsAg was screened by yeast display method. After the above screening, the inventors isolated and cloned specific single-chain antibodies scFvC (Wt) (SEQ ID NO: 1) and scFvD (Wt) with high affinity for adr and adw subtypes of HBsAg.

[0114] II. Further Screening of Mutated High Affinity scFvs

[0115] Since human monoclonal antibodies selected from human scFv yeast libraries usually have low expression levels, they are difficult to be prepared in an efficient manner and used for large-scale clinical applications. To overcome this problem, the above screened human scFvs of HBsAg specific to adr and adw subtypes (ie, scFvC and scFvD) can be further mutated. Taking s...

Embodiment 2

[0116] scFvD (wt), scFvD (1D-66) screened in Example 2 and scFvD(2D-115) can neutralize the binding of HBsAg to hepatocytes

[0117] In order to detect whether the obtained high-affinity and specific antibody can neutralize the binding of HBsAg to hepatocytes, the inventors detected the ability of the obtained single-chain antibody to specifically neutralize HBsAg. First, the inventor used 100 μl of 5 μg / ml HBsAg-bio(adw) (Shanghai PrimeGene Biotechnology Company, #671-02; in the laboratory, the purchased HBsAg(adw) was biotinylated according to the standard method, diluted to 5 μg / ml) were pre-incubated with yeast cells (5x10) expressing scFvA (A-WT), scFvD (wt), scFvD (1D-66) 6 and 1.5x10 7 cells), and the single-chain antibody scFvA (A-WT) without binding specificity was used as a negative control. The pre-incubation was carried out for 5 minutes at room temperature. With 7000rpm the yeast cells were centrifuged for 3 minutes, the supernatant was taken and mixed with...

Embodiment 3

[0119] Example 3 Monoclonal antibodies scFvD-hIgG1Fc and scFvC-hIgG1Fc can complete Total blockage of HBsAg binding to Chang hepatocytes

[0120] In order to further test whether the antibody of the present invention can prevent the binding of the virus to liver cells, according to the method described in reference 7, the inventors used the scFvC (wt) and scFvD (2D-115) screened above to construct monoclonal antibodies respectively scFvC-hIgG1Fc and scFvD-hIgG1Fc. Then, the inventors examined the ability of the scFvC-hIgG1Fc and scFvD-hIgG1Fc to neutralize the binding of HBsAg to Chang hepatocytes.

[0121] In order to detect the ability of scFvD-hIgG1Fc to neutralize the binding of HBsAg to Chang hepatocytes, Chang hepatocytes (5.3×10 5 / ml) placed in the container. Then dilute HBsAg-bio (adw) (Shanghai PrimeGene Biotechnology Company, #671-02) to 5 μg / ml. Add 25 μl of the diluted HBsAg-bio(adw) / 5ml tube to the reaction tube, then add different amounts of each antibody...

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Abstract

The present invention provides a special high affinity anti-HBV antibody, an antigen-binding fragment and a composition thereof. The present invention provides isolated nucleic acid molecule encoding the antibody or the antigen-binding fragment. The present invention further provides a drug and/or a diagnostic composition containing the antibody, the antigen-binding fragment or the antibody composition, and uses and methods of the antibody or the antigen-binding fragment for prevention, treatment and/or diagnosis of HBV infections.

Description

technical field [0001] The present invention belongs to the field of prevention and / or treatment of virus infection, especially the prevention and / or treatment of diseases caused by virus infection, such as hepatitis. In addition, the present invention also relates to the preparation and application of specific high-affinity antibodies to virus antigens. Background technique [0002] Hepatitis B virus (HBV) is a virus that causes chronic human infection. It is estimated that more than 400 million people worldwide suffer from hepatitis. HBV has antigens hepatitis B surface antigen (HBsAg), PreS1 and PreS2 1 . In China, a recent study showed that the weighted prevalence of HBsAg, anti-HBsAg, and anti-hepatitis B core (HBc) was 7.2%, 50.1%, and 34.1%, respectively, for the Chinese population aged 1-59 years 2 . Considering the low sensitivity of the HBsAg assay, the high rate of anti-HBc suggests that the actual number of infected people may be higher than initially estima...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/70C12Q1/68A61K39/42A61K48/00A61P1/16A61P31/20G01N33/577G01N33/569
CPCC12Q1/68G01N33/577C07K2317/622C12Q1/70A61K48/00C07K16/08C07K2317/565C07K2317/92C12N5/10A61K39/42G01N33/569C07K16/082C12N15/63C12N15/11A61P1/16A61P31/20
Inventor 傅阳心
Owner 傅阳心
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