Renewable synthetic antibodies for epigenomics

描述（由申请人提供）：广泛的挑战领域：（06）使能技术具体的挑战主题：06- od -106用于表观基因组研究的可再生亲和力试剂。该提案的目标是(i)建立一个生成高质量、经过良好验证的合成抗体的技术平台，以及（ii）生产用于表观遗传研究的高价值抗原的初始合成抗体。合成抗体使用最先进的体外技术生成，无需动物免疫。根据定义，它们是单克隆的，它们的表达克隆，连同它们的免疫化学特性，被永久存档并易于分发。总之，这些特性使合成抗体比单克隆和多克隆抗体优越得多。表观遗传标记（如包含特异性翻译后修饰（PTMs）的组蛋白尾部）和其他参与表观遗传调控的蛋白质的亲和试剂是表观基因组学研究中至关重要的工具。它们是染色质免疫沉淀（ChIP）的核心组成部分，ChIP是标准的、强大的工具，也是参与调节和响应表观基因组变化的蛋白质复合物的免疫组织化学和生化分析所必需的。现在非常清楚的是，传统抗体存在严重的局限性。单克隆抗体的生产缓慢且昂贵，并且对其亲和力或选择性几乎没有控制。多克隆抗体在可重复性和特异性方面有一个基本问题，这相当于不可接受的实验变化水平。这种情况表明，我们以高度质量控制的方式进行表观基因组分析的能力存在重大差距。我们的团队包括三位合成抗体工程领域的领导者和一位组蛋白修饰蛋白质组学分析领域的领导者。我们共同开发了一套完整的全重组抗体技术，其性能超过天然抗体。在这个项目中，我们将结合我们互补的专业知识，进一步增强和应用这些强大的技术，以产生对组蛋白尾部PTMs和其他参与表观遗传调控的蛋白质具有高度特异性和亲和力的合成抗体。该项目的具体目标是：(1)开发一个集成的技术平台，用于生产和表征具有精致抗原结合特性的合成抗体；(2)合成针对组蛋白尾肽和多梳成分的抗体；(3)使用多种分析工具严格验证生成的抗体用于ChIP应用；(4)开发抗体分发的网络数据库和物流基础设施。该项目产生的可再生合成抗体将对表观基因组学研究产生直接而强烈的影响，因为它们将得到高度验证，其性质（亲和力和特异性）不会改变。该项目将在快速增长的重组抗体领域雇用和培训更多的人员。总之，这个项目完全符合ARRA挑战拨款的总体目标，也符合为表观基因组研究生成可再生亲和试剂的具体挑战。组蛋白化学成分的细微修饰（翻译后修饰）是转录表观基因组调控中至关重要的生物标志物，这一过程对于理解细胞发育和疾病状态的发生至关重要。由于缺乏高质量的“亲和试剂”，可以选择性地检测和捕获组蛋白修饰和与之相关的蛋白质，这一领域的研究受到严重阻碍。该项目将建立最先进的平台，为表观基因组靶点生成高质量、可再生的亲和试剂，从而消除表观基因组研究的一个主要瓶颈。

DESCRIPTION (provided by applicant): Broad challenge area: (06) Enabling technologies Specific challenge topic: 06-OD-106 Renewable affinity reagents for epigenomic research. The goals of this proposal are (i) to establish a technology platform for generating high-quality, well- validated synthetic antibodies and (ii) to produce the initial set of synthetic antibodies to high-value antigens useful for epigenetic research. Synthetic antibodies are generated using state-of-the-art in vitro techniques without animal immunization. They are monoclonal by definition and their expression clones, together with their immunochemical properties, are permanently archived and easily distributed. Together, these attributes make synthetic antibodies much superior alternatives to monoclonal and polyclonal antibodies. Affinity reagents to epigenetic markers such as histone tails containing specific post-translational modifications (PTMs) and to other proteins involved in epigenetic regulation are critically important tools in epigenomics research. They are the central component of the chromatin immunoprecipitation (ChIP), the standard, powerful tool and also essential for immunohistochemical and biochemical analyses of protein complexes involved in regulating and responding to epigenomic changes. It is now painfully clear that conventional antibodies present severe limitations. Monoclonal antibody production is slow and expensive, and there is little control on their affinity or selectivity. Polyclonal antibodies have a fundamental problem in reproducibility and specificity, which amount to unacceptable levels of experimental variations. This situation represents a critical gap in our ability to perform epigenomic profiling in a highly quality-controlled manner. Our team includes three leaders in the field of synthetic antibody engineering and a leader in proteomic analysis of histone modifications. We have collectively developed a complete spectrum of fully recombinant antibody technologies whose performance exceeds that of natural antibodies. In this project, we will combine our complementary expertise to further enhance and apply these powerful technologies for generating synthetic antibodies of exquisite specificity and affinity to histone tail PTMs and other proteins involved in epigenetic regulation. The specific aims of this project are (1) to develop an integrated technological platform for the production and characterization of synthetic antibodies with exquisite antigen binding properties; (2) to generate synthetic antibodies to histone tail peptides and to polycomb components; (3) to rigorously validate generated antibodies for ChIP applications using multitude of analytical tools; and (4) to develop a Web- database and logistical infrastructure for antibody distribution. Renewable, synthetic antibodies generated in this project will make an immediate and strong impact on epigenomics research, because they will be highly validated and their properties (affinity and specificity) will not change. This project will employ and train additional personnel in the rapidly growing field of recombinant antibodies. Together, this project perfectly aligns with the general goal of the ARRA challenge grants and with the specific challenge of generating renewable affinity reagents for epigenomic research. Subtle modifications (post-translational modifications) in the chemical composition of histones are critically important biomarkers in the epigenomic regulation of transcription, a process that is critical for the understanding of cell development and the onset of disease states. Research in this field has been severely hindered by a lack of high- quality "affinity reagents" that can selectively detect and capture histone modifications and proteins associated with them. This project will establish a state-of-the-art platform and generate high-quality, renewable affinity reagents for epigenomic targets, thereby eliminating a major bottleneck in epigenomic research.