Anti-anthrax PA antigen monoclonal antibody and applications thereof

A monoclonal antibody, anti-anthrax technology, applied in the application field of preparing anthrax therapeutic drugs, can solve the problems of easy misdiagnosis, ineffective antibiotic treatment, missed treatment timing, etc., and achieve the effects of excellent toxin neutralization activity and good protection effect.

Active Publication Date: 2013-02-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the incubation period of anthrax infection can last for hours or even days. Symptoms at this stage are similar to those of flu, so it is easy to misdiagnose and miss the best time for treatment;
[0006] 2. The human body develops drug resistance: the abuse of antibiotics is a serious global public health problem at present, and some individuals have developed resistance to many antibiotics, making antibiotic treatment ineffective;
[0011] Chinese patent 200480017547.7 discloses a monoclonal antibody (5E8) against anthrax PA antigen, but the monoclonal antibody failed to show a good protective effect in animal experiments, so there is still a long way to go before drug development
Chinese patent 200610165844.7 also discloses a monoclonal antibody (5E1) against anthrax PA antigen, but the inventor found that at the cellular level, the binding force between 5E1 and PA is weak and cannot be used for future pharmacokinetic studies

Method used

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  • Anti-anthrax PA antigen monoclonal antibody and applications thereof
  • Anti-anthrax PA antigen monoclonal antibody and applications thereof
  • Anti-anthrax PA antigen monoclonal antibody and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1. Cloning of mouse monoclonal antibody gene

[0046] 1.1 Preparation of mouse monoclonal antibody

[0047] Using recombinant anthrax protective antigen as immunogen, a high-affinity and high-specificity mouse-derived anti-PA monoclonal antibody 5E11 hybridoma cell line was obtained by conventional monoclonal antibody preparation methods, and the subtype of the secreted antibody molecule was IgG1. The light chain is of the Kappa subtype.

[0048] 1.2 Cloning of monoclonal antibody 5E11 light and heavy chain genes

[0049] The monoclonal antibody is composed of a variable region and a constant region. Because the gene variation of the variable region of the mouse monoclonal antibody is large, and the gene sequence of the constant region is very conservative, the 5'-RACE method is used for cloning. A primer sequence was added to the 5' end, and PCR amplification was performed with the conserved sequence at the 3' end. The obtained mouse monoclonal antibody gene...

Embodiment 2

[0062] Example 2. Cloning of chimeric antibody genes

[0063] Compared with human body, murine antibody belongs to heterologous protein and has obvious anti-host antibody response when applied to human body. Therefore, the strategy of humanization transformation is often used to reduce immunogenicity. The simple and convenient method is to humanize the constant region, using human The constant region of the mouse is replaced by the constant region of the mouse, that is, the chimeric antibody is constructed.

[0064] 2.1 Codon optimization and total gene synthesis of human constant region

[0065] The expression of the protein can be improved by using optimized codons. First, the human IgG1 heavy chain constant region (GenBank accession number: Z17370) and the human Kappa light chain constant region gene (GenBank accession number: GI: 341915149) are programmed according to the mouse codon preference using software. After optimization, the optimized two gene sequences (the gene...

Embodiment 3

[0078] Example 3. Cloning of Humanized Monoclonal Antibody Gene

[0079] 3.1 Humanized monoclonal antibody gene design

[0080] The chimeric antibody replaces the mouse constant region gene with the human constant region gene, and the degree of humanization reaches 75% compared with the mouse antibody. A further method of humanization is to humanize the variable regions. The basic strategy is to keep the amino acid sequence of the complementarity determining region (CDR) unchanged, search the human antibody library sequentially for the amino acid sequence of the framework region, obtain the sequence with the greatest homology, and mutate the corresponding site.

[0081] FR1, FR2 and FR4 of the heavy chain variable region have 100% homology with gb: AAS85990.1, gb: ADW08153.1, emb: CAK50646.1, respectively, without changing the amino acid sequence. FR3 has 88% homology with emb: CAR62720.1, and there are differences in 4 amino acid positions, and site-directed mutations are p...

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Abstract

The present invention discloses an anti-anthrax PA antigen monoclonal antibody having a humanize form, a chimeric form and a murine form, and applications of the monoclonal antibody in anthrax treatment drug preparation. The anti-anthrax PA antigen monoclonal antibody has characteristics of specific antigen binding, excellent toxin neutralization activity and good animal protection function.

Description

technical field [0001] The invention relates to the gene of anti-anthrax monoclonal antibody and the polypeptide encoded by the gene, and the application of the gene and polypeptide in the preparation of anthrax therapeutic medicine. Background technique [0002] Anthrax is a major zoonotic infectious disease caused by Bacillus anthrax. Has a wide distribution worldwide. According to different routes of infection, human anthrax can be divided into three primary types: cutaneous anthrax, gastrointestinal anthrax and inhalational anthrax. In my country, pulmonary anthrax is managed as a Class I infectious disease because of its high case fatality rate. Bacillus anthracis contains two main virulence factors: a poly-D-glutamyl capsule (PGA) linked by gamma bonds and anthrax toxin composed of three protein components. The capsule can prevent bacteria from being phagocytized by host immune cells, and the toxin is the main cause of host injury and death. Anthrax toxin is encode...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N15/13C12N15/63C12N5/10A61K39/40A61K47/48A61P31/04
Inventor 陈薇李建民李冰张金龙侯利华宋小红于婷徐俊杰付玲张军刘树玲任军
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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