A Semi-Automated Antibody-Discovery Platform to Target Challenging Biomolecules

生物医学研究的一个主要瓶颈是缺乏在“真实”生物环境中直接研究疾病机制的工具，例如在生物体中，以准确的方式。我们目前对痴呆症的理解是技术差距影响的一个显著例子。痴呆症是一个总称，指的是一系列无法治愈的疾病，包括阿尔茨海默病、帕金森病和额颞叶痴呆。总的来说，这些疾病目前影响着全世界5000多万人。尽管痴呆症如此普遍，但由于对其病理机制的信息很少，我们仍然缺乏有效的诊断和治疗分子。痴呆症的一个主要机制是神经系统中蛋白质簇的形成，这与细胞死亡有关。在过去的二十年中，研究人员一直致力于在高度控制的实验条件下，使用分离的蛋白质（体外方法）来理解蛋白质聚类。这些精确的研究有助于理解调节蛋白质聚类的物理定律；然而，它们也提供了一幅过于简单的聚类机制图。他们没有解释神经系统中的许多事件，事实证明，从患者身上分离出来的蛋白质簇经过了严重的化学修饰，并与包括核酸在内的其他生物分子紧密相关。由于其与靶标的特异性结合，抗体是一类快速发展的蛋白质药物，在生物医学研究中作为探针得到了广泛的应用。抗体使科学家能够将高度精确的体外测量与高度复杂的生物样品相结合。然而，尽管具有潜力，抗体的使用仍然受到与其生产相关的挑战的阻碍。抗体的发现可能是一个漫长而昂贵的过程。此外，一些生物分子，如化学修饰的蛋白质、蛋白质簇和核酸，很难用标准的抗体发现方法靶向，尽管这些生物分子在痴呆症等疾病中非常普遍。该项目的目标是提供一种创新的、普遍适用的抗体发现技术，能够针对化学修饰的蛋白质簇或与其他生物分子（如核酸）复合的蛋白质簇。为了实现我们的目标，我们将研究两个系统，蛋白FUS和交互反应dna结合蛋白43 (TDP-43)，涉及肌萎缩性侧索硬化症，额颞叶痴呆和阿尔茨海默病。据报道，这两种蛋白质都经历了几种类型的化学修饰，并与不同的RNA分子结合。我们将使用我们的综合发现平台开发抗体，并添加半自动筛选组件，以靶向携带化学修饰和/或与疾病相关rna复合物的感兴趣蛋白质簇。因此，我们将使用抗体来监测蛋白质- rna聚集体在人体组织中的分布。我们的结果将为这些疾病提供新的信息，并导致一种普遍适用的时间和成本效益的抗体发现技术，以产生针对蛋白质以外的生物分子的抗体。

A major bottleneck in biomedical research is the scarcity of tools to study disease mechanisms directly in their 'true' biological environments, such as in living being, in an accurate manner. A remarkable example of the implications of this technology gap is given by our current understanding of dementia. Dementia is an umbrella term referring to a set of incurable diseases, including Alzheimer's, Parkinson's, and frontotemporal dementia. Altogether, these pathologies currently affect more than 50 million people worldwide. Despite this prevalence of dementia, we still lack effective diagnostic and therapeutic molecules for it because of the sparse information on the pathological mechanisms. A major mechanism of dementia is the formation of protein clusters in the nervous system, which are associated with cellular death. Over the last two decades, researchers have focused on understanding protein clustering under highly controlled experimental conditions using proteins in isolation (in vitro approaches). These accurate studies have contributed to the understanding of the physical laws that regulate protein clustering; nevertheless, they have also provided an overly simplistic picture of the clustering mechanism. They did not account for the many events in the nervous system, as proven by the fact that protein clusters isolated from patients are heavily chemically modified and tightly associated with other biomolecules, including nucleic acids.Because of their specific binding to targets, antibodies represent a fast-growing class of protein drugs and find a wide application as probes in biomedical research. Antibodies allow scientists to bridge highly precise in vitro measurements with the use of highly complex biological samples. Nevertheless, despite their potential, the use of antibodies is still hindered by challenges associated with their production. Antibody discovery can be a lengthy and costly procedure. Furthermore, several biomolecules, such as chemically modified proteins, protein clusters, and nucleic acids, are challenging to target with standard antibody-discovery approaches, despite these biomolecules being highly prevalent in diseases, e.g., dementia. The goal of this project is to deliver an innovative, generally applicable antibody-discovery technology able to target protein clusters which are chemically modified or in complex with other biomolecules, such as nucleic acids. To achieve our goal, we will work on two systems, the protein FUS and the transactive response DNA-binding protein 43 (TDP-43), involved in amyotrophic lateral sclerosis, frontotemporal dementia and Alzheimer's disease. Both proteins have been reported to undergo several types of chemical modifications and to bind to different RNA molecules. We will develop antibodies using our integrative discovery platform with the addition of a semi-automated screening component to target clusters of the proteins of interest carrying chemical modifications and/or in complex with RNAs associated with the disease. Thus, we will use the antibodies to monitor the distribution of the protein-RNA aggregates in human tissue. Our results will provide novel information on these diseases and lead to a generally applicable time- and cost-effective antibody-discovery technology to produce antibodies against biomolecules beyond proteins.