Targeted antitoxin delivery platforms as post-exposure therapies for botulism

描述（由申请人提供）：开发梭状菌神经毒素引起的肉毒中毒暴露后治疗的最大挑战之一是传统基于抗体的抗毒素的短暂治疗窗口，以中和循环中的游离神经毒素并阻止毒素与细胞受体的结合。正因为如此，在毒素已经内化，抗体无法中和之前，患者通常是无症状的。在毒素内化并出现症状后，目前的抗毒素无效。我们提出的暴露后抗毒素将解决这一关键需求。我们建议开发一种暴露后治疗性抗肉毒杆菌神经毒素（BoNT）试剂，该试剂基于抗体/抑制剂-毒素嵌合体的概念，可以靶向特异性递送到神经元细胞中，并且在进入细胞后将递送并释放抑制剂到细胞质中，从而中和BoNT的催化活性，从而使细胞能够更快地恢复。在这种嵌合抗毒素中，高亲和力的scFv抗体、基于肽的抑制剂或基于小分子的抑制剂将附着在BoNT重链（BoNT- hc）上，从而通过特异性受体结合、易位（即递送）并释放到神经元细胞的细胞质中，特异性抑制BoNT- lc的金属蛋白酶活性。使用该系统的优点是，中和抗毒素将被引导到毒素作用发生的相同位置-不仅是同一类型的细胞，而且是目标底物所在的相同细胞内隔室。重要的是，我们提出的抗毒素策略有可能逆转和/或缩短中毒（即瘫痪）的临床效应持续时间。为了达到获得这种新型暴露后抗毒素治疗的目的，我们提出以下目标：1。重组BoNT重链融合蛋白载体的构建与优化。2. 神经细胞内有效递送抑制剂的货物释放机制优化。3. 优化底物- scfv抑制剂作为中和BoNT活性的支架。4. 用于递送肽基和小分子抑制剂的替代BoNT/A-HC接头的构建和优化。

DESCRIPTION (provided by applicant): One of the greatest challenges of developing post-exposure treatments for botulism caused by clostridial neurotoxins is the brief therapeutic window for conventional antibody-based antitoxins to neutralize the free neurotoxin in the circulation and to block binding of the toxin to cellular receptors. Because of this, the patient is most often asymptomatic until after the toxin has already been internalized and is no longer accessible to antibody neutralization. No current antitoxin is effective after toxin has been internalized and symptoms manifested. Our proposed post-exposure antitoxin will address this critical need. We propose to develop a post-exposure therapeutic anti-botulinum neurotoxin (BoNT) reagent, which builds on the concept of an antibody/inhibitor-toxin chimera that can be targeted for specific delivery into neuronal cells and that upon entry into the cells will deliver and release an inhibitor into the cytosol that will neutralize the catalytic activity of BoNT, such that the cells can more rapidly recover. In this chimeric antitoxin, high affinity scFv antibodies, peptide-based inhibitors, or small molecule-based inhibitors will be attached to the heavy chain of BoNT (BoNT-HC), such that it could specifically inhibit the metalloprotease activity of BoNT-LC after specific receptor binding, translocation (i.e. delivery), and release into the cytosol of neuronal cells. The advantage of using this system is that the neutralizing antitoxin will be directed to the same location where toxin action is occurring - not only the same type of cells, but also the same intracellular compartments where the target substrates are located. Importantly, our proposed antitoxin strategy has the potential to reverse and/or shorten the duration of the clinical effects of intoxication (i.e. paralysis). To achieve the goal of obtaining this new post-exposure antitoxin therapeutic, we propose the following aims: 1. Construction and optimization of recombinant BoNT heavy chain fusion proteins as delivery vehicles. 2. Optimization of the cargo release mechanism for efficient delivery of inhibitor to the cytosol of neuronal cells. 3. Optimization of the substrate-scFv inhibitor as a scaffold for neutralization of BoNT activity. 4. Construction and optimization of alternative BoNT/A-HC adaptors for delivering peptide-based and small-molecule-based inhibitors.