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

快速发现新的生物制剂和细胞表面靶点来指导细胞行为

基本信息

批准号：
9336925
负责人：
Casim Sarkar
金额：
$ 18.25万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9336925
关键词：
Address Affinity Alpha Cell Amino Acid Sequence Ankyrin Repeat Behavior Binding Biological Assay Biomedical Research CXCR4 gene Cell Surface Receptors Cell Survival Cell membrane Cell surface Cells Clinical Complementary DNA Development Directed Molecular Evolution Disease Disseminated Malignant Neoplasm Emulsions Encapsulated Engineering Epidermal Growth Factor Receptor Fluorescence-Activated Cell Sorting Fluorescent Probes G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs Goals HIV Receptors HIV-1 Health Human In Vitro Incubated Individual Infection Knowledge Laboratories Lead Libraries Malignant Neoplasms Measures Medicine Methodology Methods Noise Oils Patients Phenotype Problem Solving Process Protein Engineering Proteins Randomized Recovery Resistance development Signal Transduction Therapeutic Time Translating Water biophysical properties cDNA Library cell behavior cell motility cellular targeting design interest novel overexpression particle receptor response scaffold screening therapeutic protein touchscreen

项目摘要

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.

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个问题，但这种生物制剂的鉴定因为不可能直接在任何感兴趣的细胞上快速筛选大的蛋白质文库以鉴定指导细胞表型的候选物。这个R21提案的目标是开发一个广泛有用的一个蛋白质工程平台，将使生物制剂的大规模表型筛选，从而从根本上改变了这些治疗方法的产生方式。这种方法需要创建一个新的体外展示方法，该方法使用非常大的天然蛋白质文库（~1011）来选择新的结合剂，特定的已知靶或整个细胞“表面组”（即，所有细胞表面靶点，包括抗肿瘤药不能从质膜功能性纯化但可能是重要临床靶点的蛋白质）。然后，该聚焦的、结合剂富集的文库（~107）将被输入到另一种新的体外展示中该方法允许根据需要直接筛选改变靶细胞表型的单个蛋白质。我们提出的方法有几个关键特征，这些特征与当前的技术水平形成了对比，该领域：1）该方法可以利用现有知识来靶向特定的细胞表面受体，可以在没有涉及所需细胞行为的靶的先前偏差的情况下应用; 2）该方法应用不可知的，并且靶细胞不必在实验室中进行工程化或生长，因此主要 3）该策略能够回收所有表面蛋白结合剂，包括那些表面蛋白结合不良的结合剂。表达的靶标或具有低结合亲和力; 4）在无偏应用中，回收的生物制剂可以用于追溯性地鉴定与表型转变有关的新的细胞表面靶标;以及5）整个方法学是快速的，因此对于可能在一种环境中进化并产生耐药性的疾病，例如癌症，患者特异性的方式，新的个性化生物制剂可以在“真实的时间”发现，以跟上疾病我们预计，这个新平台-其吞吐量的大幅增加和其效用，解决人类健康和疾病方面的广泛问题-将是变革性的，和基础生物医学研究。