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

针对翻译后修饰的高性能重组抗体的合理生成

• 批准号: 10025208
• 负责人: Takamitsu Hattori
• 金额: $ 62.04万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025208
• 关键词: Affect; Affinity; Amino Acid Sequence; Amino Acids; Antibodies; Antigens; Antineoplastic Agents; Architecture; Benchmarking; Binding; Biological Assay; Biomedical Research; Cell physiology; Chemicals; Communities; Complex; Development; Directed Molecular Evolution; Disease; Enhancers; Exhibits; Fab Immunoglobulins; Generations; Genomics; Goals; Head; Immunoglobulin G; Immunoprecipitation; Laboratories; Lead; Left; Malignant Neoplasms; Methods; Modification; Molecular; Molecular Diagnosis; Peptides; Performance; Play; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reagent; Recombinant Antibody; Recombinant Proteins; Reproducibility; Reproducibility of Results; Research; Research Personnel; Role; Series; Site; Specificity; Structure; Surface; Technology; Variant; Western Blotting; antibody engineering; anticancer research; antigen binding; cancer diagnosis; drug discovery; histone methylation; improved; innovation; innovative technologies; large datasets; molecular recognition; new technology; next generation; novel; polyclonal antibody; potential biomarker; protein function; stoichiometry; tool

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.

项目摘要 该项目的总体目标是开发有效产生高性能的强大技术 以及对翻译后修饰（PTM）的可再生抗体，并广泛制造此类试剂 可用于研究社区。 PTM是蛋白质的化学修饰，在许多人中很重要 细胞功能。 PTM的失调会导致许多疾病，包括癌症。 PTMS的抗体 是分析PTM的核心组成部分，但是许多可用的抗体具有严重的缺点，限制了 生物医学和癌症研究的进展。可用抗体的两个主要问题是低质量和 批次变化，这可能导致研究人员得出不正确的结论，并导致缺乏可重复性 在研究结果中。此外，蛋白质组学和基因组学的最新进展已使得综合 产生大型数据集的研究，社区分享了这些结果。那就是抗体问题 成为影响潜水员研究领域的全球问题。具有高特异性的抗体的产生 对PTM的高亲和力是挑战，因为它们必须区分氨基酸的微小化学变化 和密切相关的氨基酸序列。为了克服分子识别的基本困难，我们 提案是一种创新的方法，基于我们以前发现的一种独特的抗原结合模式 组蛋白甲基化的性能抗体。通常，抗原结合片段（Fab） 抗体以1：1化学计量识别其抗原。我们先前对组蛋白甲基抗体的研究 揭示了一种意外的绑定机制，我们将其称为“抗原扎实”，其中两个晶圆厂合作 通过形成头对头同型二聚体来扣紧一种抗原。抗原粘液会产生异常大的抗原 识别表面，使抗体能够获得高特异性和对PTM的高亲和力。 假设一种理性生成使用抗原扣的抗体的方法将基本上基本上 加速对PTM的高性能抗体的发展。我们的具体目的是建立一个理性的 产生抗体抗体的方法，并通过 产生对磷酸化抗原的抗体。我们将批判性地验证抗体和 基准对可用抗体进行基准测试。该项目的主要产品将是重组蛋白 定义的序列，消除了重现性的主要障碍。我们设想提出的技术和 它产生的高性能试剂将对PTM及其进行更强大和彻底的分析 在癌症等疾病中的作用。