Producing Polyclonal Human Antibodies to Anthrax

描述(申请人提供)：炭疽病是一种高风险的生物恐怖威胁。尽管接受了大剂量的抗生素治疗，但在2001年的邮件袭击事件后，仍有几人死亡。用抗炭疽免疫球蛋白被动免疫，结合抗生素，是一种很有前途的暴露后治疗方法。血液科技公司正在开发一种转基因牛，这种牛将生产出人类免疫球蛋白而不是牛免疫球蛋白。这些牛可以用炭疽抗原进行高度免疫，并产生高效的多克隆抗体，以防止炭疽和许多其他生物恐怖分子的攻击。在第一阶段，我们将含有人免疫球蛋白重链和轻链基因全序列的人人工染色体导入牛成纤维细胞，并利用体细胞克隆技术获得了跨染色体(TC)牛。结果表明，TC小牛保留了HAc，并在血液中产生了低水平的人免疫球蛋白(2-30 mg/L)。我们还在野生型荷斯坦牛身上开发了一种免疫方案，使用各种抗原佐剂配方和加强疫苗来产生针对炭疽的高滴度抗体。此外，我们还建立和优化了检测炭疽保护性抗原、水肿性因子和致死性因子抗原的抗体效价的ELISA法，并改进了体外毒素中和试验，以定量和证明抗体的生物学效力。在第二阶段的支持下，我们将为第一阶段生产的TC牛接种疫苗和加强免疫，收集血浆并分别纯化人和牛的抗体。我们将改进体内小鼠保护试验，并与纯化的牛抗体相比，评估纯化的人抗体的治疗效力。我们还将培育牛重链基因被敲除的TC小牛，以努力提高人类免疫球蛋白的产生水平。第二阶段的最终目标是完成牛系统的开发，以生产对治疗炭疽感染有效的人类多克隆抗体。

DESCRIPTION (provided by applicant): Anthrax is a high risk bioterrorist threat. Despite high dose antibiotic therapy, several deaths occurred following the 2001 mail attack. Passive immunization with anti-anthrax immunoglobulins, in combination with antibiotics, is a promising approach for post-exposure treatment. Hematech is developing a genetically modified line of cattle that will produce human instead of bovine immunoglobulin. These cattle may be hyperimmunized with anthrax antigens and produce highly efficacious polyclonal antibodies for protection against anthrax, and many other bioterrorist agents. In phase I, we introduced a human artificial chromosome containing the entire sequences of human immunoglobulin heavy and light chain genes into bovine fibroblasts and produced transchromosomic (Tc) cattle using somatic cell cloning technology. We showed that Tc calves retain the HAC and produce low levels of human IgG in blood (2-30 mg/L). We also developed an immunization scheme, in wild wildtype Holstein steers, using various antigen-adjuvant formulations and booster vaccinations to produce high titer antibodies to anthrax. Furthermore, we developed and optimized ELISA assays, which were used to detect IgG titers to anthrax protective antigen, edema factor and lethal factor antigens, and we refined an in-vitro toxin neutralization assay, to quantify and demonstrate the biological potency of the antibodies. Under Phase II support we will vaccinate and boost Tc cattle produced in Phase I, collect plasma and purify human and bovine antibodies separately. We will refine an in vivo mouse protection assay and evaluate the therapeutic potency of the purified human antibody as compared to purified bovine antibody. We will also produce Tc calves in which the bovine heavy chain genes have been knocked out in an effort to increase levels of human immunoglobulin production. The ultimate goal of phase II is to complete the development of a bovine system for production of human polyclonal antibodies with efficacy in the treatment of anthrax infections.