利用转基因小鼠筛选全人源炭疽致死因子中和抗体

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

摘要:

炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分：保护性抗原 (PA)、致死因子 (LF) 和水肿因子 (EF)。PA与LF形成致死毒素 (LT)，与EF形成水肿毒素 (ET)。由于致死毒素 (LT) 在感染者损伤及死亡中发挥主要作用，因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效，治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体，美国FDA已批准瑞西巴库 (人源PA单抗) 用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变，针对PA单一表位的抗体将可能失效，因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体，而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠，利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞，通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体，通过毒素中和实验 (TNA) 发现1D7和2B9在细胞模型中均显示较好的中和活性，并且与PA单抗联合使用时，表现出较好的协同作用。总之，本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势，快速筛选到全人源LF抗体，为快速筛选全人源单克隆抗体开辟了新的思路与方法。

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.