Rapid discovery of new biologics and cell-surface targets to direct cell behavior

医学上的一个重大挑战是在不操纵细胞的情况下控制细胞的行为 在内部。蛋白质疗法有可能解决这一问题，但此类生物制品的鉴定 仍然很困难，因为不可能直接在任何感兴趣的细胞上快速筛选大型蛋白质文库 以确定决定细胞表型的候选基因。此R21提案的目标是开发一种广泛有用的 蛋白质工程平台，将实现对生物制剂的大规模表型筛选，从而从根本上 改变了这些疗法的创建方式。这种方法需要在体外创造一种新的 使用极大的天然蛋白质文库(~1011)来选择新的结合剂的展示方法 既可以是特定的已知靶标，也可以是整个细胞的表面体(即所有细胞表面的靶标，包括顽固者 不能从质膜上进行功能性纯化但可能成为重要临床靶点的蛋白质)。 然后，这个聚焦的、富含结合剂的文库(~107)将被输入到另一个新的体外展示中 允许直接筛选可根据需要改变目标细胞表型的单个蛋白质的方法学。 我们建议的方法有几个关键特征，它们与 该领域：1)该方法可以利用现有的知识来靶向特定的细胞表面受体，或者它 可以在没有涉及所需细胞行为的目标的先验偏差的情况下应用；2)该方法是 应用不可知的细胞和目标细胞不需要在实验室中设计或培养，因此主要 可以使用细胞；3)该策略能够回收所有表面粘合剂，包括那些较差的粘合剂 表达的靶标或具有低结合亲和力的；4)在无偏见的应用中，回收的生物制品可以 用于追溯识别与表型转变有关的新的细胞表面靶点；以及5) 整体方法是快速的，因此对于癌症等可能在 以患者特定的方式，可以实时发现新的个性化生物制剂，以跟上 疾病。我们预计，这一新平台将大幅提升吞吐量，并在 解决人类健康和疾病的广泛问题-将对翻译和 和基础生物医学研究。

A grand challenge in medicine is harnessing control over the behavior of a cell without manipulating it internally. Protein therapeutics have the potential to solve this problem, but identification of such biologics remains difficult because it is not possible to rapidly screen large protein libraries directly on any cell of interest to identify candidates that direct cell phenotype. The goal of this R21 proposal is to develop a broadly useful protein engineering platform that will enable large-scale phenotypic screening of biologics, thus fundamentally transforming the way in which these therapeutics are created. This approach entails creation of a new in vitro display methodology that uses extremely large naïve protein libraries (~1011) to select for novel binders to either a specific known target or the entire cell ‘surfaceome’ (i.e., all cell-surface targets, including recalcitrant proteins that cannot be functionally purified from the plasma membrane but may be important clinical targets). Then, this focused, binder-enriched library (~107) will be the input into another new in vitro display methodology that allows direct screening of individual proteins that alter target cell phenotype as desired. There are several key features of our proposed methodology that contrast it with the current state of the art in the field: 1) the approach can either leverage existing knowledge to target a specific cell-surface receptor or it can be applied without prior bias of targets implicated in the desired cell behavior; 2) the approach is application-agnostic and the target cells do not have to be engineered or grown in the laboratory, so primary cells can be used; 3) the strategy enables recovery of all surfaceome binders, including those to poorly expressed targets or with low binding affinities; 4) in unbiased applications, the recovered biologics can be used to retroactively identify novel cell-surface targets that are implicated in the phenotype transition; and 5) the overall methodology is rapid, so for diseases such as cancer that may evolve and develop resistance in a patient-specific manner, new personalized biologics could be discovered in ‘real time’ to keep pace with the disease. We anticipate that this new platform – with its massive increase in throughput and its utility in addressing a broad range of issues in human health and disease – will be transformative to both translational and fundamental biomedical research.