A kind of bispecific recombinant anti-hbsag antibody, its preparation method and use

A bispecific antibody and carrier technology, which is applied in the field of prevention, bispecific recombinant anti-HBsAg antibody, and treatment of hepatitis B virus infection, can solve the problems of not meeting clinical needs, high development costs, and limiting the development process, etc.

Active Publication Date: 2017-11-10
SHENZHEN SCIPROGEN BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the multifactorial nature of the disease, single-target therapy can no longer meet clinical needs
There have been reports of preclinical experiments on the combined use of two monoclonal antibodies in clinical trials, but this combined use of monoclonal antibodies has many limitations [Kou G, Shi J, Chen L, et al. A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin. Cancer Lett 2010;299:130-6]
For example, the safety and efficacy of the combined application of two monoclonal antibodies need to be evaluated separately. The resulting long cycle and high development costs limit the development process. Bispecific antibodies just solve this bottleneck[Wu C, Ying H, Grinnell C, et al.Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 2007;25:1290-7]

Method used

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  • A kind of bispecific recombinant anti-hbsag antibody, its preparation method and use
  • A kind of bispecific recombinant anti-hbsag antibody, its preparation method and use
  • A kind of bispecific recombinant anti-hbsag antibody, its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of HBsAg-specific single B cells

[0041] By separating lymphocytes from the peripheral blood of volunteers who had been injected with hepatitis B vaccine, HBsAg was separated with CD19-PE / Cy5, IgG-PE, HBsAg-biotin, Streptavidin-FITC fluorescent antibodies + CD19 + IgG + B cells. Such as figure 1 As shown, a small population of HBsAg-positive cells (approximately 0.01-0.02% of the entire B cell population) can be seen. Each HBsAg sorted by MoFlo XDP ultrafast flow cytometry sorting system + CD19 + IgG + B cells were injected into 96-well Single cell PCR plates, ensuring one cell per PCR well. Then, the lysate was added, frozen in liquid nitrogen, and stored in ice at -80°C for later use.

Embodiment 2

[0042] Embodiment 2: RT-PCR and nested PCR clone antibody variable region gene and gene expression

[0043] Take the 96-well Single cell PCR plates frozen in a -80°C refrigerator, thaw slowly on ice, and then add reverse transcriptase to synthesize cDNA. Since the cDNA synthesized by a single cell is extremely rare, the antibody gene contained in it must be amplified by nested PCR. After two rounds of PCR, obvious PCR band amplification (about 400bp in size) can be seen. The paired PCR bands of the light and heavy chains were recovered and sequenced. Such as figure 2 As shown, after analyzing its sequence, specific PCR amplifies the variable region band of the antibody, and introduces restriction enzyme cutting sites. The PCR product of the amplified variable region of the antibody was connected with the constant region of the antibody and then inserted into the expression vector pcDNA3.1(+).

Embodiment 3

[0044] Example 3: Monoclonal Antibody Gene Expression

[0045] Paired antibody light chain and heavy chain plasmids were transiently transfected into Freestyle 293F cells cultured in serum-free medium, and the cell supernatant was collected 7 days after transfection, and the IgG antibody in the cell supernatant was purified by Protein A. A total of 42 anti-HBsAg monoclonal antibody strains were obtained. SDS-PAGE electrophoresis identification such as image 3 As shown, the purified IgG antibody shows two bands on 10% SDS-PAGE, one of which is about 50KD (heavy chain), and the other is about 25KD (light chain).

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Abstract

The invention belongs to the field of biotechnology and discloses a bispecific antibody specifically binding to different epitopes of hepatitis B surface antigen (HBsAg). The present invention also provides the amino acid sequence of the antibody, the DNA sequence encoding the antibody, the expression vector containing the DNA sequence and the host cell containing the expression vector. The invention also discloses the preparation method of the above-mentioned antibody and its application in preventing hepatitis B virus infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a bispecific recombinant anti-HBsAg antibody, its preparation method and its application in preventing and treating hepatitis B virus infection. Background technique [0002] Hepatitis B virus (HBV) is a double-stranded DNA virus that causes chronic hepatitis B, leading to liver failure, cirrhosis and liver cancer. There are approximately 350 million HBV-infected people in the world, and this has become a world public health problem. In recent decades, the successful development of hepatitis B vaccine has brought great hope to the prevention of hepatitis B. However, there are indeed a small number of people who receive hepatitis B vaccine but cannot stimulate the immune system to produce anti-hepatitis B antibodies. Once this part of the population is exposed to high-risk factors for hepatitis B, they can only be prevented by injection of human hepatiti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C07K16/08C12N15/13C12N15/85C12N5/10A61K39/42A61P31/20
Inventor 钱卫珠谈文龙
Owner SHENZHEN SCIPROGEN BIO PHARMA
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