Development of De-immunized Botulinum Neurotoxin Type A for Dystonia

描述（由申请人提供）：本修订的探索/开发（R21）提案描述了消除肌张力障碍患者对A型肉毒杆菌神经毒素（BoNT/A）治疗的免疫反应的创新研究方法。BoNT/A的免疫原性产生中和抗体，其减少或消除其治疗益处。这项拟议的研究源于这样一种认识，即蛋白质疗法的发展通常必须由对药物诱导的免疫反应的洞察来驱动。功能性治疗剂去免疫化的可推广策略将消除或至少减少“基因到药物”的快速翻译的这一主要障碍。这种新方法将尖端的生物信息学工具与经典的免疫学测定相结合，以创建一种改进的BoNT/A，其以与肿瘤细胞和病毒相同的方式逃避免疫学检测。目标将是抗体产生的T细胞依赖性机制，其由负载肽的II类MHC-T细胞受体相互作用介导。该项目的主要目标是使用表位作图算法来计算鉴定BoNT/A衍生的T细胞表位。随后的验证将使用从BoNT/A暴露患者抽取的血液的MHC结合测定和T细胞刺激研究进行实验。免疫显性表位将被靶向修饰以降低其免疫原性，目的是保留毒素的总体折叠。将使用计算方法来验证修饰毒素的免疫原性降低，并使用同源性建模来确定这些变化的结构效应。将测定去免疫肽序列的MHC结合以及体外和体内（小鼠）T细胞刺激。公共卫生相关性：了解A型肉毒杆菌神经毒素的免疫原性，将有助于改善肌张力障碍治疗的发展。

DESCRIPTION (provided by applicant): This revised Exploratory/Developmental (R21) proposal describes innovative research approaches to eliminate the immune response to botulinum neurotoxin type A (BoNT/A) therapy in dystonia patients. The immunogenicity of BoNT/A gives rise to neutralizing antibodies that reduce or eliminate its therapeutic benefits. The proposed research stems from a recognition that development of protein therapeutics in general must be driven by insights into drug-induced immune responses. A generalizable strategy for de- immunization of functional therapeutics will eliminate or at least reduce this major obstacle to the rapid translation of "genes to drugs". This novel approach combines cutting-edge bioinformatics tools with classic immunological assays to create an improved BoNT/A that escapes immunological detection much in the same way as tumor cells and viruses. The target will be the T cell dependent mechanism for antibody generation, which is mediated by peptide-loaded class II MHC-T cell receptor interactions. The primary goals of the project are to use epitope mapping algorithms to computationally identify BoNT/A- derived T cell epitopes. Subsequent verification will be performed experimentally using MHC binding assays and T cell stimulation studies of blood drawn from BoNT/A exposed patients. Immunodominant epitopes will be targeted for modification to reduce their immunogenicity with the aim of preserving the overall fold of the toxin. Computational methods will be used to verify reduced immunogenicity of the modified toxin, and homology modeling will be used to determine the structural effects of these changes. The de-immunized peptide sequences will be assayed for MHC binding and in vitro and in vivo (mouse) T cell stimulation. Public Health Relatedness: Knowledge of the elements of botulinum neurotoxin type A that give rise to its immunogenicity will contribute to the development of improved dystonia therapy.