Novel Carbohydrate-binding Antibodies to Human Glycans Using the Lamprey System

使用 Lamprey 系统开发针对人类聚糖的新型碳水化合物结合抗体

• 批准号: 10672258
• 负责人: RICHARD D CUMMINGS
• 金额: $ 49.91万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672258
• 关键词: Address; Affinity; Amino Acid Sequence; Animals; Antibodies; Antibody Formation; Antibody Specificity; Antigens; Binding; Biological Sciences; CD Antigens; Carbohydrates; Cell Line; Cell surface; Cells; Chimera organism; Collection; Communities; Complementary DNA; Complex; Computer Analysis; Coupling; DNA; Development; Disease; Engineering; Ensure; Expression Library; Fishes; Generations; Genomics; Glycoengineering; Glycolipids; Glycopeptides; Glycoproteins; Health; Homeostasis; Human; Human Biology; Hybridomas; Immune Sera; Immune system; Immunization; Immunize; Immunologic Techniques; Immunologics; Immunology; Individual; Interferometry; Lampreys; Lectin; Libraries; Lymphocyte; Mammals; Maps; Methods; Mining; Modeling; Molecular; Monoclonal Antibodies; Monosaccharides; Mus; Mutagenesis; Paraffin Embedding; Petromyzon marinus; Plants; Polysaccharides; Process; Proteins; Protocols documentation; Reagent; Recombinants; Reproducibility; Research; Research Personnel; Role; Shotguns; Sialic Acids; Source; Specificity; Structure; Sulfate; Surface; System; Technology; Tissues; Yeasts; antigen binding; cell fixing; functional group; human disease; human tissue; innovation; innovative technologies; metabolomics; murine monoclonal antibody; novel; receptor; response; screening; success; technology development; tool; transcriptomics; virtual screening

Summary This application addresses a major unmet need for technology that will facilitate the integration of glycomics and glycan expression with genomics, transcriptomics, and metabolomics. The rapidly developing field of glycoscience must have tools to identify and characterize the expression of human and animal glycans in the context of normal development, homeostasis, and in disease processes. Traditional studies in the glycosciences have relied on an odd assortment of plant-derived lectins and a few traditional mouse monoclonal antibodies, but such reagents have multiple limitations. This application exploits our breakthrough technology that allows us to develop suites of specific, rigorously characterized anti-carbohydrate antibodies (ACAs). We have developed high-throughput immunization and screening technologies using variable-lymphocyte receptors (VLRBs) that are generated upon immunization of ancient sea lampreys with cells, cell-conjugates, glycoproteins, or tissues. Because of the evolutionary diversity of both their immune systems and glycomes as compared to mammals, these ancient fish generate a diverse repertoire of anti-carbohydrate VLRBs with exquisite specificity, and are able to discriminate between different linkages, motif presentation on N- and O-glycans, and the presence of functional groups on individual monosaccharides. After immunization, the cloned cDNAs encoding the VLRBs are expressed in a yeast surface display (YSD) library containing multiple individual specificities. This innovative technology development allows us to both generate a ‘library of VLRBs’ in a single immunization protocol, as well as sequentially screen these stable YSD libraries for ACAs using our numerous and diverse glycan and glycopeptide microarrays and display technologies. Ultimately, after the VLRBs are screened for their binding to glycan antigens, recombinant and reproducible VLRB-Ig chimeras are expressed, which can be used for all types of immunological approaches to explore glycan expression and function. We propose 3 robust and innovative Specific Aims to address the current technological deficiencies: Aim 1- Refine and expand our technology to generate permanent VLRB YSD libraries against a diverse array of human glycan targets, using multiple immunization and screening strategies. Aim 2- Enrich for and characterize monoclonal VLRBs that are specific for human glycans, which will be critical in mapping the Human Glycome, by mining the created YSD libraries. Aim 3- Generate novel VLRB specificities using targeted mutagenesis of characterized VLRBs, to create tailored antibody specificities with potentially higher affinities. The success of our studies will climax with an arsenal of rigorously vetted tools and robust reagents that will be made publicly available and accessible to the broader research community. This will ultimately encourage widespread study of these incredibly important, complex molecules that are vital in human health and disease, promote harmonization with other biological sciences, and allow for the study of expression and functions of the Human Glycome.

概括 该应用解决了对促进糖组学和糖组学整合的技术的主要未满足需求。 聚糖表达与基因组学、转录组学和代谢组学的快速发展领域。 糖科学必须有工具来识别和表征人类和动物聚糖的表达 正常发育、稳态和疾病过程的背景。 依赖于各种奇怪的植物源凝集素和一些传统的小鼠单克隆抗体， 但此类试剂有多种局限性。该应用利用了我们的突破性技术。 开发一套特定的、经过严格表征的抗碳水化合物抗体（ACAs）。 使用可变淋巴细胞受体（VLRB）的高通量免疫和筛选技术 通过用细胞、细胞结合物、糖蛋白或组织对古代七鳃鳗进行免疫而产生。 由于与哺乳动物相比，它们的免疫系统和糖组具有进化多样性， 这些古老的鱼类产生了多种具有精致特异性的抗碳水化合物 VLRB，并且 能够区分 N- 和 O- 聚糖上的不同连接、基序呈现以及 免疫后，克隆的 cDNA 编码 VLRB。 在包含多种个体特异性的酵母表面展示（YSD）文库中表达。 技术开发使我们能够在单一免疫方案中生成“VLRB 库”，如 以及使用我们众多且多样化的聚糖和序列筛选这些稳定的 YSD 文库中的 ACA 最终，筛选 VLRB 的结合后，进行糖肽微阵列和显示技术。 表达聚糖抗原、重组且可重复的VLRB-Ig嵌合体，可用于所有类型 我们提出了 3 种稳健且创新的免疫学方法来探索聚糖表达和功能。 解决当前技术缺陷的具体目标： 目标 1 - 完善和扩展我们的技术 使用多种方法针对多种人类聚糖靶标生成永久 VLRB YSD 文库 免疫和筛选策略 目标 2- 富集并表征特异性单克隆 VLRB。 通过挖掘创建的 YSD 库，对人类聚糖进行分析，这对于绘制人类聚糖图谱至关重要。 目标 3 - 使用特征化 VLRB 的定向诱变产生新的 VLRB 特异性，以创建定制的 具有潜在更高亲和力的抗体特异性将随着一系列的研究而达到高潮。 经过严格审查的工具和强大的试剂将公开提供给更广泛的人使用 这最终将鼓励研究界对这些极其重要、复杂的领域进行广泛研究。 对人类健康和疾病至关重要的分子，促进与其他生物科学的协调，以及 允许研究人类糖组的表达和功能。

项目成果

Reusable glycan microarrays using a microwave assisted wet-erase (MAWE) process.

使用微波辅助湿擦除 (MAWE) 过程的可重复使用的聚糖微阵列。

• 发表时间: 2024-03-26
• 影响因子: 4.3
• 作者: Mehta, Akul Y;Tilton, Catherine A;Muerner, Lukas;von Gunten, Stephan;Heimburg;Cummings, Richard D
• 通讯作者: Cummings, Richard D