Producing Polyclonal Human Antibodies to Anthrax

描述（由申请人提供）：炭疽是一种高风险的生物恐怖主义威胁。尽管进行了大剂量抗生素治疗，但在2001年邮电袭击之后仍发生了几起死亡事件。使用抗炭疽免疫球蛋白进行被动免疫，并结合抗生素，是一种很有前途的暴露后治疗方法。Hematech正在开发一种转基因牛，这种牛将生产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超免疫，并产生高效的多克隆抗体，以保护其免受炭疽和许多其他生物恐怖制剂的侵害。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，利用体细胞克隆技术培育出了转染色体体牛。我们发现Tc小牛保留了HAC，并在血液中产生低水平的人IgG （2-30 mg/L）。我们还开发了一种免疫方案，在野生的野生型荷斯坦牛中，使用各种抗原-佐剂配方和加强疫苗来产生高滴度的炭疽抗体。此外，我们开发并优化了ELISA检测方法，用于检测炭疽保护抗原、水肿因子和致死因子抗原的IgG滴度，并改进了体外毒素中和试验，以量化和证明抗体的生物效力。在第二阶段的支持下，我们将为第一阶段生产的Tc牛接种和增强疫苗，收集血浆并分别纯化人抗体和牛抗体。我们将完善体内小鼠保护试验，并评估纯化的人抗体与纯化的牛抗体的治疗效力。为了提高人类免疫球蛋白的产量，我们还将生产母牛重链基因被敲除的Tc小牛。II期的最终目标是完成牛系统的开发，用于生产有效治疗炭疽感染的人多克隆抗体。

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.