Immunotherapeutics to Prevent & Treat Botulinum Toxin Intoxication

描述(由申请人提供)：在被确认为疾控中心A类潜在武器或高优先级制剂的制剂中，有肉毒杆菌毒素(BONT)。由于其极强的效力和杀伤力、易于生产和运输以及需要对受影响的人进行长期的重症护理，BONT构成了一个重大的生物武器威胁。这项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的行为。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。建议进行实验以确定人上皮细胞运输BONT所需的序列。我们将首先确定极化的人上皮细胞摄取所需的最小毒素片段。结合和跨细胞作用都将被测量。定点突变将被用于进一步定位结合、跨细胞或两者兼而有之所需的毒素上的位置。来自肺和胃肠道的极化上皮细胞系将被用来确定跨越不同上皮的通道是否需要相同的毒素结构域。将产生针对负责上皮结合和跨细胞作用的区域(或亚区域)的单抗。将测试单抗抑制结合和/或跨细胞作用的能力。细胞内转运途径和用于跨细胞转运的上皮细胞上的受体(S)都将被确定。如果一种蛋白质在受体中起作用，它就会被克隆。抗体同型和效价在抑制BONT跨上皮转运中的作用将被确定。单抗的可变区将被克隆并表达为IgA，它是多聚体，因此具有更强的亲和力，以及分泌型IgA(SLGA)，它带有共价连接的分泌片，也是聚合体，但对蛋白酶更具抵抗力。效应器功能激活和价态在免疫球蛋白背景下的作用也将被调查。将努力生产一种基于抗体的蛋白质，这种蛋白质可以进入神经元的胞浆，并中和细胞内的BONT。将产生有效中和BONT催化活性的单抗。我们将尝试将它们传递到神经元的细胞质中，方法是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，或者用中和单链抗体的scFv版本取代BONT的催化域。

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.