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: 10293635
• 负责人: RICHARD D CUMMINGS
• 金额: $ 50.4万
• 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/10293635
• 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; Fc Receptor; Fishes; Generations; Genomics; 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; base; 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 该应用程序解决了对技术的主要未满足需求，这将有助于糖的整合和 基因组学，转录组学和代谢组学的聚糖表达。快速发展的领域 糖科学必须具有识别和表征人类和动物聚糖表达的工具 正常发育，稳态和疾病过程中的背景。糖基镜的传统研究 依靠各种植物来源的讲座和一些传统的小鼠单克隆抗体， 但是，这样的试剂有多个限制。该应用程序利用了我们的突破性技术，使我们 开发特定，严格特征的抗碳水化合物抗体（ACA）的套件。我们已经发展了 使用可变淋巴细胞受体（VLRB）的高通量免疫抑制和筛查技术 与细胞，偶联物，糖蛋白或组织免疫抑制的免疫抑制产生。 由于其免疫系统和甘氨酸的进化多样性与哺乳动物相比 这些古老的鱼产生了具有独特特异性的抗碳水化合物VLRB的潜水员曲目，并且是 可以区分不同的链接，n-和o-glycans的图案表现以及存在 单个单糖的官能团。免疫后，编码VLRB的克隆的cDNA 在包含多个个人规格的酵母表面显示（YSD）库中表达。这种创新 技术开发使我们能够在单个免疫抑制协议中生成“ VLRB库”，作为 以及依次使用我们众多和多样的聚糖和 糖肽微阵列和展示技术。最终，将VLRB筛选为结合到 表达了聚糖抗原，重组和可再现的VLRB-gig嵌合体，可用于所有类型 探索聚糖表达和功能的免疫学方法。我们提出了3个强大而创新的 具体目的旨在解决当前的技术缺陷：目标1-完善并将我们的技术扩展到 使用多种 免疫和筛查策略。 AIM 2-富集并表征特定的单克隆VLRB 对于人类的聚糖而言，通过挖掘创建的YSD库来绘制人类的聚糖至关重要。 AIM 3-使用特征VLRB的靶向诱变生成新颖的VLRB规格，以创建量身定制 抗体规格可能具有较高的亲和力。我们的研究的成功将以武器库为高潮 严格审查的工具和健壮的试剂将公开可用，可以更广泛地使用 研究社区。这最终将鼓励对这些非常重要，复杂的宽度研究 在人类健康和疾病中至关重要的分子，促进与其他生物科学的协调，以及 允许研究人类糖的表达和功能。