Technology Development for Recombinant Affinity Reagents

我们建议建立一个研究合作，在其中我们改进目前用于产生亲和力试剂的筛选和评估技术。我们打算改进发现过程，使其不仅比目前可能的成本更低、速度更快，而且还能生产出比目前的单抗更通用、更有用的试剂。该中心将涉及布赖恩·凯博士(芝加哥伊利诺伊大学)、安德烈亚斯·普鲁克顿博士(苏黎世大学)和迈克尔·韦纳博士(LLUMINA公司)的实验室。我们将重点介绍三种类型的重组亲和试剂：人单链可变区片段(ScFv)、设计的Ankyrin重复蛋白(DARPins)和纤维连接蛋白III型(FN3)单体。ALED Edwards博士(结构基因组学中心)将为我们提供100个目标，我们将把我们的技术改进应用到这些目标上。我们还将使用与一组测试蛋白质的N-末端和C-末端相对应的合成肽作为目标。我们的研究计划包括三个战略目标。首先，通过分子工程，我们将产生多功能的亲和试剂，这些试剂可以完成抗体的许多标准任务，以及作为细胞内靶标的生物传感器和诱导抑制物的功能。其次，我们计划通过使用乳剂来改进我们目前的选择协议，使它们更快、更小、更便宜、更好。第三，除了我们自己的表征外，我们还将向五个Beta测试者分发亲和试剂，用于评估它们在生物应用中的应用，并收到关于应该在亲和试剂中设计哪些额外特性的反馈。一旦我们准备并审查了高质量的亲和试剂，它们将通过亚利桑那州立大学的DNA储存库和两家试剂供应公司提供。我们研究的首要目标将是优化能够以通用、强大、成本效益和高通量的方式应用于整个人类蛋白质组的技术。

We propose to form a research collaboration in which we improve upon the screening and evaluation technologies currently used to generate affinity reagents. We intend to refine the discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. This Center will involve the laboratories of Drs. Brian Kay (University of Illinois at Chicago), Andreas Pluckthun (University of Zurich), and Michael Weiner (lllumina Corporation). We will focus on three types of recombinant affinity reagents: human single-chain Fragments of variable regions (scFv), Designed Ankyrin Repeat Proteins (DARPins), and fibronectin type III (FN3) monobodies. Dr. Aled Edwards (Structural Genomics Center) will supply us with 100 targets to which we will apply our technology improvements. We will also use synthetic peptides, corresponding to the N-termini and C-termini of a test set of proteins, as targets. Our research plan consists of three strategic goals. First, through molecular engineering we will generate versatile affinity reagents that can accomplish the many standard tasks of antibodies, as well as function as biosensors and inducible inhibitors of intracellular targets. Second, we plan to improve our current selection protocols by using emulsions to make them faster, smaller, cheaper, and better. Third, in addition to our own characterization, we will distribute affinity reagents to five beta-testers for their evaluation in biological applications and receive feedback on what additional properties should be engineered into the affinity reagents. Once we have prepared and vetted high quality affinity reagents, they will be made available through the DNA repository at Arizona State University and two reagent supply companies. The overarching goal of our research will be to optimize technologies that can be applied to the entire human proteome in a versatile, powerful, cost-effective, and high-throughput manner.