A Cell-Penetrating Anthrax Antitoxin

描述(申请人提供)：这项研究的目标是开发一种经济、安全、有效的治疗方法，以逆转炭疽芽孢杆菌毒素(致命毒素)的催化成分Lf在中毒细胞的细胞质中的活性。成功实现这一目标将带来比目前使用抗生素或抗毒素更好的结果。 像raxibacumab或PBI-220，在炭疽杆菌生物恐怖袭击的情况下，这可以拯救无数人的生命。我们建议筛选人源化的骆驼文库，以鉴定高亲和力的单链可变区，一个纳米体，能够阻断蛋白酶活性的致死因子(LF)。然后，我们将创建阻止LF的纳米体与另一个具有转运活性的LF，LFN结构域的基因融合，但缺乏催化活性，使其能够在PA存在的情况下运输到哺乳动物细胞的细胞质中。因此，我们计划增选炭疽杆菌毒素输送系统，转而将中和纳米体输送到中毒细胞中。 该项目涉及两个研究所的两个研究小组，全景研究所(PRI)和行星生物技术研究所(PBI)。在PRI，生物素化的Lf将被用来筛选人源化的骆驼文库，并鉴定具有高亲和力并能够阻断Lf的蛋白酶活性的克隆。它们还将被选中，以确保它们不会与LFN结合，LFN是用于将它们转移到细胞内的结构域。 我们将对纳米体进行修饰，使其具有LFN结构域的融合蛋白，使其能够以PA依赖的方式转移到细胞内，并能够到达中毒细胞胞浆中的LF。我们将使用快速瞬时植物表达系统表达纳米体融合蛋白，并使用亲和层析对融合蛋白进行纯化。首先，我们将测试它们进入细胞的能力，然后测量它们抑制中毒细胞中LF酶活性的能力。

DESCRIPTION (provided by applicant): The goal of this research is to develop an economical, safe, effective therapy to reverse the activity of the catalytic component, LF, of the Bacillus anthracis toxin (lethal toxin), within the cytoplasm of the intoxicated cell. Success in achieving this goal will lead to better outcomes than currently seen with antibiotics or antitoxins like raxibacumab or PBI-220, and this could save countless more lives in the event of a bioterrorist attack with B. anthracis. We propose to screen the humanized camelid library to identify high-affinity single chain variable fragment, a nanobody, able to block protease activity of lethal factor (LF). We will then create genetic fusion of the nanobody that blocks LF with another domain of LF, LFN, that had the translocating activity buts is devoid of catalytic activity to enable it to be transported into the cytoplasm of mammalian cells in the presence of PA. Thus, we plan to co-opt the B. anthracis toxin delivery system to deliver, instead, a neutralizing nanobody into intoxicated cells. This project involves two research teams at two institutes, Panaorama Research Institute (PRI) and Planet Biotechnology (PBI). At PRI, biotinylated LF will be used to screen the humanized camelid library and identify clones that have high affinity and are ability to block protease activty of LF. They will also be selected to ensure that they do not bind LFN, the domain that will be used to translocate them into the cell. We will modify the nanobody to make fusion proteins with the LFN domain, giving it the ability to be translocated into cells, in a PA-dependent manner, and be able to reach LF in the cytoplasm of intoxicated cells. We will express the nanobody fusion proteins using the rapid transient plant expression system and purify the fusions using affinity chromatography. First, we will test their ability to enter cells and subsequently measure their ability to inhibit LF enzymatic activity in intoxicated cells.