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强大的技术，有效地产生高性能 和翻译后修饰（PTM）的可再生抗体，并广泛地制备这样的试剂 提供给研究界。PTM是蛋白质的化学修饰，在许多生物学领域都很重要。 细胞功能。PTM的失调导致许多疾病，包括癌症。PTM抗体 是分析PTM的核心成分，但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， 批间差异，这可能导致研究人员得出错误的结论，并导致缺乏重现性 在研究成果中。此外，蛋白质组学和基因组学的最新进展使全面的 产生大型数据集的研究，社区分享这些结果。因此，抗体问题具有 成为影响不同研究领域的世界性问题。高特异性抗体的产生 对PTM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小化学变化， 和密切相关的氨基酸序列。为了克服分子识别中的基本困难，我们 提出了一种创新的方法，建立在我们以前发现的一种独特的抗原结合模式的高， 组蛋白甲基化的抗体。常规地，免疫球蛋白的抗原结合片段（Fab）是免疫球蛋白的抗原结合片段（Fab）。 抗体以1：1的化学计量识别其抗原。我们以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，我们称之为“抗原抱持”，其中两个Fab协同作用， 通过形成头对头同源二聚体来扣紧一个抗原。抗原结合产生了非常大的抗原- 这使得抗体能够实现对PTM的高特异性和高亲和力。我们 假设使用抗原抱合合理产生抗体方法将基本上 加速开发针对PTM的高性能抗体。我们的具体目标是建立一个理性的 方法产生的抱合抗体，并证明我们的方法的广泛适用性， 产生针对磷酸化抗原的抱合抗体。我们将严格验证抱合抗体， 将其与现有抗体进行比对。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，所提出的技术和 它生产的高性能试剂将使PTM及其 在癌症等疾病中的作用。