Rational generation of high-performance recombinant antibodies to post-translational modifications

项目总结 该项目的总体目标是开发高效地产生高性能的强大技术 和可再生的翻译后修饰抗体(PTM)，并广泛地制造此类试剂 可供研究界使用。PTM是蛋白质的化学修饰，在许多 细胞功能。PTMS的失调会导致许多疾病，包括癌症。抗PTMS抗体 是分析PTM的中心成分，但许多可用的抗体都有严重的缺点，限制了 生物医学和癌症研究的进展。现有抗体的两个主要问题是质量低和 批次之间的差异，这可能会导致研究人员得出错误的结论，并导致缺乏重复性 在研究结果中。此外，蛋白质组学和基因组学的最新进展使 产生大型数据集的研究，社区共享这些结果。因此，抗体问题有 成为影响各个研究领域的世界性问题。高特异性抗体的产生 而且对PTMS的高亲和力是具有挑战性的，因为它们必须区分氨基酸中的微小化学变化 和密切相关的氨基酸序列。为了克服分子识别的根本困难，我们 在我们之前发现的一种独特的抗原结合模式的基础上，提出了一种创新的方法 抗组蛋白甲基化的性能抗体。传统上，疫苗的抗原结合片段(Fab) 抗体以1：1的化学计量比识别其抗原。我们先前对组蛋白甲基化抗体的研究 揭示了一种意想不到的结合机制，我们将其称为“抗原结合”，其中两个Fabs协同工作 通过形成头对头的同源二聚体来结合一个抗原。抗原结合产生了非常大的抗原- 识别表面，使抗体能够实现对PTMS的高特异性和高亲和力。我们 假设一种使用抗原结合合理产生抗体的方法将实质上 加快研制抗PTMS的高性能抗体。我们的具体目标是建立一种理性的 产生结合抗体的方法，并通过以下方式展示我们方法的广泛适用性 产生针对磷酸化抗原的紧贴抗体。我们将严格验证紧贴抗体和 以可用的抗体为基准。该项目的主要产品将是重组蛋白，具有 明确的序列，消除了重复性的主要障碍。我们设想，拟议的技术和 其生产的高性能试剂将使对PTMS及其 在癌症等疾病中的作用。

PROJECT SUMMARY The overall goals of this project are to develop powerful technology that efficiently generates high-performance and renewable antibodies to post-translational modifications (PTMs), and to make such reagents broadly available to the research community. PTMs are chemical modifications of proteins that are important in many cellular functions. Dysregulation of PTMs contributes to many diseases, including cancer. Antibodies to PTMs are a central component in analyzing PTMs, but many available antibodies have severe shortcomings, limiting the progress of biomedical and cancer research. Two major issues with available antibodies are low quality and lot-to-lot variation, which could lead researchers to incorrect conclusions and contribute to a lack of reproducibility in research results. Moreover, recent advances in proteomics and genomics have enabled comprehensive studies that produce large datasets, and the community shares those results. Thus, the antibody problem has become a world-wide problem affecting diverse research fields. The generation of antibodies with high specificity and high affinity to PTMs is challenging, because they must discriminate small chemical changes in amino acids and closely related amino acid sequences. To overcome fundamental difficulties in molecular recognition, we propose an innovative approach built on our previous discovery of a unique antigen-binding mode of high- performance antibodies to histone methylation. Conventionally, the antigen-binding fragment (Fab) of an antibody recognizes its antigen with 1:1 stoichiometry. Our previous studies of antibodies to histone methylation revealed an unexpected binding mechanism, which we dubbed “antigen clasping”, where two Fabs cooperatively clasp one antigen by forming head-to-head homodimers. Antigen clasping creates exceptionally large antigen- recognition surfaces, which enables antibodies to achieve high specificity and high affinity to PTMs. We hypothesize that an approach to rationally generate antibodies that use antigen clasping will substantially accelerate the development of high-performance antibodies to PTMs. Our specific aims are to establish a rational approach for generating clasping antibodies, and to demonstrate the broad applicability of our approach by generating clasping antibodies to phosphorylated antigens. We will critically validate clasping antibodies and benchmark them against available antibodies. Primary products of this project will be recombinant proteins with defined sequences, eliminating a major barrier to reproducibility. We envision that the proposed technology and the high-performance reagents it produces will enable more robust and thorough analyses of PTMs and their roles in diseases such as cancer.