Immunotherapeutics to Prevent & Treat BoNT Intoxication

描述（由申请人提供）：在CDC A类或高优先级制剂中被确定为潜在武器的制剂是肉毒杆菌毒素（BoNT）。肉毒杆菌毒素是一种主要的生物武器威胁，因为它具有极强的效力和致命性，易于生产和运输，而且需要对受影响的人进行长期的特别护理。该提案的目标是开发基于免疫的疗法，以对抗将BoNT用作生物恐怖主义武器。由于BoNT不能穿过完整的皮肤，当用作生物武器时，预计BoNT将通过胃肠道（GI）、呼吸道或两者的上皮细胞进入。实验提出，以确定什么样的序列所需的BoNT运输由人类上皮细胞。我们将首先确定跨极化的人上皮细胞摄取所需的最小毒素片段。将测量结合和转胞吞。定点突变将用于进一步定位毒素上结合、转胞吞作用或两者所需的位点。源自肺和胃肠道的极化上皮细胞系将用于确定跨不同上皮的通路是否需要相同的毒素结构域。将产生针对负责上皮结合和转胞吞作用的结构域（或亚结构域）的单克隆抗体（mAb）。将检测mAb抑制结合和/或转胞吞作用的能力。将鉴定细胞内运输途径和上皮细胞上用于转胞吞作用的受体。如果一个蛋白质在受体中起作用，它将被克隆。将确定抗体同种型和效价在抑制BoNT跨上皮转运中的作用。 来自mAb的可变区将被克隆并表达为伊加，其是聚合的，因此表现出增强的亲合力，以及表达为具有共价连接的分泌片段的分泌型伊加（slgA），其也是聚合的，但对蛋白酶更具抗性。还将研究效应子功能激活和效价在IgG背景下的作用。将努力生产一种基于抗体的蛋白质，该蛋白质可以进入神经元的胞质溶胶并中和细胞内的BoNT。将产生有效中和BoNT催化活性的mAb。我们将尝试通过将它们与识别神经元表面分子并被内吞的抗体进行基因融合，或者通过用中和mAb的scFv版本替换BoNT的催化结构域，将它们递送到神经元的细胞质中。

DESCRIPTION (provided by applicant): Among agents identified as potential weapons under CDC category A, or high priority agents, is botulinum toxin (BoNT). BoNT poses a major bioweapon threat because of its extreme potency and lethality, its ease of production and transport, as well as the need for prolonged intensive care among affected persons. The goal of this proposal is to develop immune-based therapeutics to counter the use of BoNT as a weapon of bioterrorism. Since BoNT cannot cross intact skin, when used as a bioweapon, it is expected that BoNT would gain entry via epithelial cells of the gastrointestinal (GI), respiratory tract or both. Experiments are proposed to determine what sequences are required for BoNT transport by human epithelial cells. We will first determine the minimal toxin fragment required for uptake across polarized human epithelial cells. Both binding and transcytosis will be measured. Site-directed mutation will be used to further map the site on the toxin required for binding, transcytosis or both. Polarized epithelial cell lines originating from both the lung and the GI tract will be used to determine if access across different epithelia requires the same toxin domain. Monoclonal antibodies (mAbs) will be produced to the domain (or sub-domain) responsible for epithelial binding and transcytosis. The mAbs will be tested for their ability to inhibit binding and/or transcytosis. Both the intracellular trafficking pathway and the receptor(s) on the epithelium used for transcytosis will be identified. If a protein functions in the receptor, it will be cloned. The role of antibody isotype and valence in the inhibition of BoNT transport across the epithelium will be determined. Variable regions from mAbs will be cloned and expressed as IgA, which is polymeric and hence exhibits enhanced avidity, and as secretory IgA (slgA) with covalently attached secretory piece, which is also polymeric but is more resistant to proteases. The role of effector function activation and valence in the context of IgG will also be investigated. Efforts will be made to produce an antibody-based protein that can gain access to the cytosol of the neuron and neutralize intracellular BoNT. mAbs effective in neutralizing the catalytic activity of BoNT will be produced. We will attempt to deliver these into the cytoplasm of neurons either by genetically fusing them to antibodies that recognize surface molecules of neurons and are endocytosed or by replacing the catalytic domain of BoNT with a scFv version of the neutralizing mAb.