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Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice

Xiaolin Wang1 Xiangyang Chi1 Ju Liu1 Weicen Liu1 Shuling Liu1 Shunfang Qiu1 Zhonghua Wen1 Pengfei Fan1 Kun Liu1 Xiaohong Song1 Ling Fu1 Jun Zhang1 Changming Yu1

(1.Institute of Biotechnology, Academy of Military Medical, Beijing, China 100071)

【Abstract】Anthrax is a highly lethal infectious disease caused by the spore-forming bacterium Bacillus anthracis. The major virulence factor of B. anthracis consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA binds with LF to form lethal toxin (LT), and PA binds with EF to form edema toxin (ET). Antibiotics is hard to work in advanced anthrax infections, because injuries and deaths of the infected are mainly caused by lethal toxin (LT). Thus, the therapeutic neutralizing antibody is the most effective treatment of anthrax. Currently most of the anthrax toxin antibodies are monoclonal antibodies (MAbs) for PA and US FDA has approved ABTHRAX humanized PA monoclonal antibody for the treatment of inhalational anthrax. Once B. anthracis was artificially reconstructed or PA had mutations within recognized neutralization epitopes, anti-PA MAbs would no longer be effective. Therefore, anti-LF MAbs is an important supplement for anthrax treatment. Most of the anti-LF antibodies are murine or chimeric antibodies. By contrast, fully human MAbs can avoid the high immunogenicity of murine antibodies. First, we used LF to immunize the transgenic mice and used fluorescent cell sorting to get antigen-specific memory B cells from transgenic mice spleen lymphocytes. By single cell PCR method, we quickly found two strains of anti-LF MAbs with binding activity, 1D7 and 2B9. Transiently transfected Expi 293F cells to obtain MAbs protein after purification. Both 1D7 and 2B9 efficiently neutralized LT in vitro, and had good synergistic effect when mixed with anti-PA MAbs. In summary, combining the advantages of transgenic mice, fluorescent cell sorting and single-cell PCR methods, this study shows new ideas and methods for the rapid screening of fully human monoclonal antibodies.

【Keywords】 anthrax lethal factor; transgenic mice; fluorescent cell sorting; memory B cells; single cell PCR; fully human MAbs; anthrax lethal factor; transgenic mice; fluorescent cell sorting; memory B cells; single cell PCR; fully human MAbs;

【DOI】

【Funds】 National Natural Science Foundation of China (No. 81172980) National Natural Science Foundation of China(No.81172980) National High Technology Research and Development Program of China (863 Program) (No. 2014AA021407) National High Technology Research and Development Program of China(863 Program)(No.2014AA021407)

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 11, Pages 1590-1599

November 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • References