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.

医学中的一个巨大挑战是利用对细胞行为的控制而无需操纵内部。蛋白质理论具有解决此问题的潜力，但是鉴定了这种生物制剂仍然很困难，因为不可能直接在任何感兴趣的细胞上迅速筛选大型蛋白质库确定直接细胞表型的候选者。该R21提案的目标是开发一个广泛有用的蛋白质工程平台将实现生物制剂的大规模表型筛查，因此从根本上讲改变创建这些治疗剂的方式。这种方法需要创建新的体外使用极大的幼稚蛋白质文库（〜1011）为新颖的粘合剂选择到的显示方法要么是特定的已知目标或整个单元格“ Surfaceome”（即所有细胞表面靶标，包括顽固的靶标无法从质膜中纯化的蛋白质，但可能是重要的临床靶标）。然后，这个聚焦，富含粘合剂的库（〜107）将是另一个新的体外显示器的输入方法可以直接筛选根据需要改变靶细胞表型的单个蛋白质。我们提出的方法论有几个关键特征，它们与当前的艺术状态进行了对比领域：1）该方法可以利用现有知识来靶向特定的细胞表面受体或IT 可以在所需的细胞行为中实现的目标事先偏差时应用； 2）方法是应用程序不合时式和目标细胞不必在实验室中进行工程或生长，因此可以使用细胞； 3）该策略能够恢复所有Surfaceome粘合剂，包括较差的策略表达的靶标或低结合亲和力； 4）在公正的应用中，回收的生物制剂可以是用于追溯识别与表型转变有关的新型细胞表面靶标； 5）总体方法是迅速的特定于患者的方式，可以在“实时”中发现新的个性化生物制剂，以使空间与疾病。我们预计这个新平台 - 随着吞吐量的大量增加及其实用性解决人类健康和疾病中广泛的问题 - 将是转化的转化和基本生物医学研究。