Proteome Scale Multiplexed Generation of Recombinant Antibodies

项目摘要/摘要。 抗体在生物科学中是一种无价的工具，越来越多地被用作治疗方法。超过了 在过去的几年里，发展了大量的蛋白质组学技术和大规模的研究来探索 生物和生化过程的规模在不断扩大。这些方法中的许多都受到以下限制 优质抗体试剂的可用性，仍在为每个抗原靶点和 缺乏可用的抗体序列。此外，还提出了对质量和 目前商品化抗体的重复性。虽然已经描述了高通量方法能够 产生针对数百种抗原的抗体是缓慢的、费力的，通常需要大量的 自动化，并依赖于抗原的可用性。这些问题，加上日益增长的对 许多高质量的抗体，凸显了迫切需要开发能够进行蛋白质组级抗体的方法 一代。 在这里，我介绍了一项激进的建议，即创建一个平台，可以产生针对所有潜在抗体的抗体 以目标蛋白质组中的抗原为靶标，然后进行蛋白质组规模的交叉反应测试。 这种方法将在人类和斑马鱼蛋白质组上进行演示，包括三个部分。 首先，我将开发基因合成技术来创建蛋白质组规模的基因库，该库由数十个 成千上万的抗原。第二，我将通过改变多样性的用途来产生高多样性的抗体库 产生逆转录元件以在体内进行靶向突变。这将允许我们绕过多样性 通常由转型造成的瓶颈。第三，我将开展大规模的图书馆对图书馆的遴选 使用多重蛋白质片段互补分析进行体内抗体-抗原相互作用。这个 产生的抗体将针对所有抗原进行重新筛选，以测量它们的交叉反应性。这项研究将 将抗体产生的成本降低至少两个数量级，并导致快速产生 高特异性、序列验证抗体的大型文库，鉴于这些抗体普遍存在的性质 试剂，将对基础和临床研究的所有领域产生重大影响。此外， 在这里概述的基因合成和体内靶向突变的技术进展将具有广泛的意义 适用于生命科学研究的许多领域。

Project Summary/Abstract. Antibodies are an invaluable tool in the biological sciences and are increasingly used as therapeutics. Over the past few years a large number of proteomic techniques and large scale studies have been developed to probe biological and biochemical processes at ever increasing scales. Many of these methods have been limited by the availability of quality antibody reagents which are still generated one-by-one for each antigen target and the lack of antibody sequences available. Additionally, large concerns have been raised regarding the quality and reproducibility of current commercial antibodies. While high-throughput methods have been described capable of generating antibodies against hundreds of antigens, they are slow, laborious, usually require significant automation, and are dependent on antigen availability. These issues, combined with the growing need for many quality antibodies, highlight the urgent need to develop methods capable of proteome-scale antibody generation. Here I introduce a radical proposal to create a platform which can generate antibodies against all potential antigen targets in a target proteome and subsequently carry out proteome-scale tests of cross-reactivity. This approach will be demonstrated on the human and zebrafish proteomes and consists of three parts. First, I will develop gene synthesis technologies to create proteome-scale gene libraries consisting of tens of thousands of antigens. Second, I will generate high diversity libraries of antibodies by repurposing diversity generating retroelements to carry out targeted mutagenesis in-vivo. This will allow us to bypass the diversity bottlenecks normally imposed by transformation. Third, I will carry out a large scale library-on-library selection for antibody-antigen interactions in-vivo using a multiplexed protein fragment complementation assay. The resulting antibody hits will be rescreened against all antigens to measure their cross-reactivity. This study will reduce the cost of antibody generation by at least two-orders of magnitude and lead to the rapid creation of large libraries of high-specificity, sequence-verified antibodies which, given the ubiquitous nature of these reagents, will have a significant impact on all areas of basic and clinical research. Furthermore, the technological developments in gene-synthesis and targeted in-vivo mutagenesis outlined will here have broad applicability in many areas of life sciences research.