Human Monoclonal Antitoxin to Anthrax Protective Antigen

描述(申请人提供)：来自几个动物模型的数据以及在人类中的轶事证据表明，如果及时给予治疗，针对炭疽保护性抗原(PA)的抗体(Abs)是有保护作用的。不幸的是，这种抗体的大量和稳定来源没有储存起来，以备炭疽芽孢杆菌的生物恐怖袭击；然而，制造抗PA单抗(MAbs)的细胞系将为这种试剂提供一个现成的来源。虽然已经描述了啮齿动物抗PA单抗，但没有一种在动物模型中显示出对完全毒力菌株的保护作用，尽管其中一种确实导致豚鼠死亡时间的显著延迟。这些负面结果可能是由于使用了不能中和毒素的表位特异性单抗和/或需要使用两个或两个以上抗毒素单抗的组合，以协同作用防止炭疽毒素的致命影响。也有可能的是，小鼠B细胞对PA的反应不能维持对参与毒素中和的表位的强烈反应。生产和使用人类单抗毒素将有许多好处：根据定义，它们是完全人类的，因此可以在没有工程技术的情况下用作人类的治疗试剂。此外，它们代表了针对炭疽的保护性人类多克隆抗体反应，因此将提供有关可用于开发分子疫苗的表位和抗体模拟表位的信息，这将比目前正在使用或正在开发的疫苗更有效。出于这些原因，我们建议进行实验，以制备和鉴定抗炭疽PA的人源单抗。因此，在目标1中，我们提出了制造人源性抗PA单抗的杂交瘤。这些细胞将从人类志愿者的外周血液中产生，这些志愿者是由重组PA(RPA)组成的实验性疫苗的接受者。最后一次免疫后约1周从接种者处获取细胞，用EB病毒转化，培养并筛选产生抗PA抗体。抗体阳性孔中的细胞将与异种骨髓瘤系融合，反复筛选反应性，并以有限稀释进行克隆，直到实现单克隆性。在目标2中，将对制备的各种单抗进行研究，以确定它们所识别的表位，以及特异性、亲和力和免疫球蛋白亚型与保护活性的关系。在体外，将通过测量单个单抗或单抗组合阻断炭疽毒素致死效应的能力来评估保护功能。

DESCRIPTION (provided by applicant): Data from several animal models, as well as anecdotal evidence in humans, suggest that antibodies (Abs) to the protective antigen (PA) of anthrax are protective if administered in a timely manner. Unfortunately, a large and stable source of such Abs is not available to stockpile for use in case of a bioterrorist attack with Bacillus anthracis; however, cell lines making anti-PA monoclonal antibodies (mAbs) would provide a ready source for such reagents. While rodent anti-PA mAbs have been described, none has shown protection in animal models against fully virulent strains, although one did lead to a significant delay in guinea pigs in time to death. These negative results may be attributable to the use of mAbs specific for epitopes that do not neutralize the toxins and/or to the need to use a combination of two or more anti-toxin mAbs that would act synergistically to protect against the lethal effects of anthrax toxins. It is also possible that the mouse B cell repertoire against PA does not sustain a strong response against epitopes that are involved in toxin neutralization. Production and use of human monoclonal antitoxins would have many advantages: they are, by definition, completely human and are therefore usable without engineering as therapeutic reagents in humans. Moreover, they are representative of the protective human polyclonal Ab response against anthrax and would therefore provide information about the epitopes and Ab mimotopes that could be used to develop molecular vaccines, which would be more efficacious than those currently in use or in development. For these reasons, we propose experiments to produce and characterize human mAbs against PA of anthrax. Thus, in Aim 1 we propose to produce heterohybridomas making human anti-PA mAbs. These will be produced from the peripheral blood of human volunteers who are recipients of an experimental vaccine consisting of recombinant PA (rPA). Cells from vaccinees will be obtained ~1 week after the final immunization, transformed with Epstein-Barr virus, cultured and screened for anti-PA production. Cells in Ab-positive wells will be fused with a heteromyeloma line, repeatedly screened for reactivity, and cloned at limiting dilution until monoclonality is achieved. In Aim 2, the various mAbs made will be studied to determine the epitopes, which they recognize, and how specificity, affinity and IgG subtype correlate with protective activity. Protective functions will be assessed in vitro by measuring the ability of individual mAbs or combinations of mAbs to block the lethal effects of anthrax toxin.