Screening of full human anthrax lethal factor neutralizing antibody in transgenic mice
【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;
 Leppla SH, Robbins JB, Schneerson R, et al. Development of an improved vaccine for anthrax. J Clin Invest, 2002, 110(2): 141–144.
 Holty JEC, Bravata DM, Liu H, et al. Systematic review: a century of inhalational anthrax cases from 1900 to 2005. Ann Intern Med, 2006, 144(4): 270–280.
 Workgroup P. Biological and chemical terrorism: strategic plan for preparedness and response. MMWR, 2000, 49: 1–14.
 Liu S, Crown D, Miller-Randolph S, et al. Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo. Proc Natl Acad Sci USA, 2009, 106(3): 12424–12429.
 Hicks CW, Cui X, Sweeney DA, et al. The potential contributions of lethal and edema toxins to the pathogenesis of anthrax associated shock. Toxins, 2011, 3(9): 1185–1202.
 Chen Z, Moayeri M, Purcell R. Monoclonal antibody therapies against anthrax. Toxins, 2011, 3(8): 1004–1019.
 Saylor C, Dadachova E, Casadevall A. Monoclonal antibody-based therapies for microbial diseases. Vaccine, 2009, 27: G38–G46.
 Chow SK, Casadevall A. Monoclonal antibodies and toxins: a perspective on function and isotype. Toxins, 2012, 4(6): 430–454.
 Peterson JW, Comer JE, Noffsinger DM, et al. Human monoclonal anti-protective antigen antibody completely protects rabbits and is synergistic with ciprofloxacin in protecting mice and guinea pigs against inhalation anthrax. Infect Immun, 2006, 74(2): 1016–1024.
 Migone TS, Subramanian GM, Zhong J, et al. Raxibacumab for the treatment of inhalational anthrax. New Engl J Med, 2009, 361(2): 135–144.
 Lim NK, Kim JH, Oh MS, et al. An anthrax lethal factor-neutralizing monoclonal antibody protects rats before and after challenge with anthrax toxin. Infect Immun, 2005, 73(10): 6547–6551.
 Little SF, Leppla SH, Friedlander AM. Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin. Infect Immun, 1990, 58(6): 1606–1613.
 Staats HF, Alam SM, Scearce RM, et al. In vitro and in vivo characterization of anthrax anti-protective antigen and anti-lethal factor monoclonal antibodies after passive transfer in a mouse lethal toxin challenge model to define correlates of immunity. Infect Immun, 2007, 75(11): 5443–5452.
 Zhao P, Liang X, Kalbfleisch J, et al. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Hum Antibodies, 2002, 12(4): 129–135.
 Kulshreshtha P, Bhatnagar R. Inhibition of anthrax toxins with a bispecific monoclonal antibody that cross reacts with edema factor as well as lethal factor of Bacillus anthracis. Mol Immunol, 2011, 48(15): 1958–1965.
 Brossier F, Lévy M, Landier A, et al. Functional analysis of Bacillus anthracis protective antigen by using neutralizing monoclonal antibodies. Infect Immun, 2004, 72(11): 6313–6317.
 Chen Z, Moayeri M, Crown D, et al. Novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody. Infect Immun, 2009, 77(9): 3902–3908.
 Pohl MA, Rivera J, Nakouzi A, et al. Combinations of monoclonal antibodies to anthrax toxin manifest new properties in neutralization assays. Infect Immun, 2013, 81(6): 1880–1888.
 Steinitz M. Three decades of human monoclonal antibodies: past, present and future developments. Hum Antibodies, 2008, 18(1-2): 1–10.
 Tomizuka K, Shinohara T, Yoshida H, et al. Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and κ loci and expression of fully human antibodies. Proc Natl Acad Sci USA, 2000, 97(2): 722–727.
 Smith K, Garman L, Wrammert J, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc, 2009, 4(3): 372–384.
 Liao HX, Levesque MC, Nagel A, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods, 2009, 158(1): 171–179.
 Chi X, Li J, Liu W, et al. Generation and characterization of human monoclonal antibodies targeting anthrax protective antigen following vaccination with a recombinant protective antigen vaccine. Clin Vaccine Immunol, 2015, 22(5): 553–560.
 Sun ZW, Wang S, Chen HP. Application of tramsgenic mice in the antibody drug denelopment. Biotechnol Busin, 2013(6): 21–25 (in Chinese).
 Morris L, Chen X, Alam M, et al. Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting. PLoS ONE, 2011, 6(9): e23532.