RAPID: Rapid Prototyping and Manufacturing of Polyclonal Anti-Ebola Antibodies with Synthetic Biology and Microbioreactors

为了对抗目前的埃博拉疫情，一种治疗方案包括一种名为“ZMapp”的三种抗体的混合物，这种抗体已被证明可以治愈灵长类动物，据报道，在目前的埃博拉疫情中，它已被用于治疗少数人。尽管ZMapp的前景光明，但由于它的可用性严重有限，它的使用一直高度限制在少数患者中。这种低可用性是因为ZMapp的生产目前是在工厂进行的，这是一个缓慢的过程，很难迅速扩大。其他抗埃博拉抗体同样受到生产系统的限制，这些生产系统在设计和扩大规模方面具有挑战性、耗时且昂贵。因此，我们迫切需要一种可通用的平台，这种平台可以快速设计以生产抗感染药物，然后很容易地扩大规模以生产大量剂量。在这个快速方案中，优化了快速生产抗感染抗体的酵母菌株将被用于表达抗埃博拉抗体。酵母是制造治疗性分子的有希望的宿主，因为它们可以在不冷藏的情况下运输，快速生长到大规模，并经过修饰以制造人性化治疗。预计工程酵母将用于经济和大规模生产抗埃博拉疗法。此外，这项工作将为应对未来新出现的疾病提供快速反应能力，因为酵母平台可以用合成生物学工具快速设计以产生新的治疗剂。此外，将采用分布式生物制造方法，将工程酵母与一种新型微生物反应器技术结合起来，这种技术有可能在疾病暴发的源头上建立快速反应。该RAPID提案的目标是在毕赤酵母中建立一个快速和灵活的生物制造平台，用于生产抗埃博拉中和抗体。ZMapp是一种由3种中和单克隆抗体（mab）组成的鸡尾酒，已被证明在感染后5天内给药可100%拯救恒河猴。Zmapp1目前是在植物烟叶（Nicotiana benthamiana）中生产的，这是一个缓慢且难以规模化的过程。目前，从其他宿主（如CHO细胞）中表达中和抗体的努力也需要大量的时间和昂贵的基础设施来扩大规模。此外，在最易受埃博拉疫情影响的欠发达地区，快速交付和长期储存单克隆抗体疗法和埃博拉疫苗可能会很困难。最后，病毒突变可能需要快速开发新的治疗药物。因此，一个可快速设计和可部署的平台，用于便携式和可扩展生产多种抗感染疗法，将有助于应对当前的埃博拉危机以及未来的传染病暴发。在这个项目中，巴斯德酵母菌株将被设计用于生产抗埃博拉单克隆抗体。pastoris可以用人类糖基化途径进行工程改造，可以冻干，并且在分泌生物制剂和单克隆抗体方面效率很高，从而实现工业规模生产和简化纯化。该小组已经用合成生物学工具改造了帕斯德梭菌，以实现生产生物药物的菌株的快速和特异性工程。通过将改造过的帕斯德酵母菌株与便携式微生物反应器相结合，将实现抗埃博拉单抗的分布式和便携式生产。该技术还可以使用合成生物学工具和高度适应性的生物制造平台快速生成和扩大新的治疗分子，以测试和部署针对不断发展的病毒和新出现的感染。

1511431 Lu, Timothy Massachusetts Institute of Technology To counter the current Ebola outbreak one treatment option involves a cocktail of three antibodies called 'ZMapp', which has been shown to cure primates and has been reportedly used in the current outbreak to treat a small number of people. Despite the promise of ZMapp, its use has been highly restricted to just a few patients because of its severely limited availability. This low availability is because ZMapp production is currently carried out in plants, a slow process that is difficult to expand rapidly. Other anti-Ebola antibodies are similarly constrained by production systems that are challenging, time-consuming, and expensive to engineer and scale-up. Thus, there is a tremendous need for generalizable platforms that can be rapidly engineered to produce anti-infectious drugs and then easily scaled-up to create large numbers of doses. In this RAPID proposal yeast strains optimized for rapid production of anti-infectious antibodies will be adapted to express anti-Ebola antibodies. Yeast are promising hosts for manufacturing therapeutic molecules because they can be transported without refrigeration, quickly grown to large scales, and modified to make humanized therapies. It is anticipate that the engineered yeasts will be useful for economical and large-scale manufacturing of anti-Ebola therapies. In addition, this work will provide rapid-response capabilities for tackling future emerging diseases because the yeast platform can be quickly engineered with synthetic biology tools to generate new therapeutic agents. In addition, a distributed biomanufacturing approach will be applied by coupling engineered yeast with a novel micro-bioreactor technology that has the potential to mount rapid responses at the source of disease outbreaks.The goal of this RAPID proposal is to establish a rapid and flexible biomanufacturing platform in Pichia pastoris for the production of anti-Ebola neutralizing antibodies. ZMapp, a cocktail of 3 neutralizing monoclonal antibodies (mAbs), has been shown to rescue 100% of rhesus macaques when administered up to 5 days post-infection. Zmapp1 is currently produced in the plant Nicotiana benthamiana, a slow process that is difficult to scale. Current efforts to express neutralizing antibodies from other hosts, such as CHO cells, also require substantial time and expensive infrastructure to scale. Moreover, quick delivery and long-term storage of mAb therapies and Ebola vaccines will likely be difficult in the under-developed areas most susceptible to Ebola outbreaks. Finally, viral mutations may necessitate the rapid development of new therapeutic agents. Thus, a rapidly engineerable and deployable platform for the portable and scalable production of multiple anti-infectious therapies would be useful for addressing the current Ebola crisis as well as future infectious outbreaks. In this project P. pastoris strains will be designed to produce anti-Ebola mAbs. P. pastoris can be engineered with human glycosylation pathways, can be lyophilized, and is highly efficient at secreting biologics and mAbs, thus enabling industrial-scale production and simplifying purification. The group has already modified P. pastoris with synthetic-biology tools to achieve rapid and specific engineering of strains that manufacture biologic drugs. Distributed and portable production of anti-Ebola mAbs will be achieved by integrating engineered P. pastoris strains with portable micro-bioreactors. The technology also enables new therapeutic molecules to be rapidly generated and scaled-up for testing and deployment against evolving viruses and emerging infections using synthetic biology tools and a highly adaptable biomanufacturing platform.