Human Monoclonal Antitoxin to Anthrax Protective Antigen

描述（由申请人提供）：来自几种动物模型的数据以及人类的轶事证据表明，如果及时给予炭疽保护性抗原（PA）的抗体（Abs）具有保护作用。不幸的是，这种抗体没有大量稳定的来源来储存，以备炭疽芽孢杆菌的生物恐怖袭击时使用；然而，制造抗pa单克隆抗体（mab）的细胞系将为这些试剂提供现成的来源。虽然啮齿类动物抗pa单克隆抗体已被描述，但没有一种单克隆抗体在动物模型中显示出对完全毒力菌株的保护作用，尽管一种单克隆抗体确实导致豚鼠死亡时间的显著延迟。这些阴性结果可能是由于使用了针对不能中和毒素的表位的特异性单克隆抗体和/或需要使用两种或两种以上抗毒素单克隆抗体的组合，这些单克隆抗体将协同作用以防止炭疽毒素的致命影响。也有可能小鼠B细胞库对PA没有维持对参与毒素中和的表位的强烈反应。人类单克隆抗毒素的生产和使用将具有许多优点：根据定义，它们完全属于人类，因此无需工程就可作为人类治疗试剂使用。此外，它们代表了针对炭疽的保护性人类多克隆抗体反应，因此将提供有关表位和抗体半模位的信息，可用于开发分子疫苗，这将比目前使用或开发的疫苗更有效。由于这些原因，我们提出了实验，以产生和表征人类单克隆抗体对抗炭疽杆菌。因此，在Aim 1中，我们提出制备制备人抗pa单克隆抗体的异杂交瘤。这些疫苗将从接受重组PA （rPA）实验性疫苗的人类志愿者的外周血中产生。最终免疫1周后，从疫苗接种者身上获得细胞，用eb病毒转化，培养和筛选抗pa生产。抗体阳性细胞将与异骨髓瘤细胞系融合，反复筛选反应性，并在有限稀释下克隆，直到获得单克隆。在Aim 2中，将研究制备的各种单克隆抗体，以确定它们识别的表位，以及特异性、亲和力和IgG亚型如何与保护活性相关。通过测量单个单克隆抗体或单克隆抗体组合阻断炭疽毒素致死效应的能力，将在体外评估保护功能。

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.