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

总结 该申请解决了对促进糖组学和生物学的整合的技术的主要未满足的需求。 聚糖表达与基因组学、转录组学和代谢组学。快速发展的领域 糖科学必须有工具来识别和表征人类和动物聚糖在 在正常发育、体内平衡和疾病过程中。糖科学的传统研究 依赖于一种奇怪的植物源凝集素和一些传统的小鼠单克隆抗体， 但这些试剂具有多种局限性。这个应用程序利用了我们的突破性技术， 开发一套特异性的、严格表征的抗碳水化合物抗体（ACA）。我们已经开发 使用可变淋巴细胞受体（VLRB）的高通量免疫和筛选技术， 在用细胞、细胞缀合物、糖蛋白或组织免疫古海七鳃鳗后产生。 由于与哺乳动物相比，它们的免疫系统和糖组的进化多样性， 这些古老的鱼类产生了多种具有精确特异性的抗碳水化合物VLRB， 能够区分不同的连接，N-和O-聚糖上的基序呈现，以及 单个单糖上的官能团。免疫后，克隆编码VLRB的cDNA， 在含有多种个体特异性的酵母表面展示（YSD）文库中表达。这一创新 技术发展使我们能够在单一免疫方案中产生“VLRB文库”， 以及使用我们的众多和不同的聚糖顺序筛选这些稳定的YSD文库中的ACA， 糖肽微阵列和显示技术。最后，在筛选VLRB的结合后， 聚糖抗原，重组和可重复的VLRB-Ig嵌合体表达，其可用于所有类型 免疫学方法来探索聚糖的表达和功能。我们提出3个稳健和创新的 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，针对不同的人聚糖靶点生成永久性VLRB YSD文库， 免疫和筛查战略。目的2-富集和表征特异性单克隆VLRB 对于人类聚糖，这将是至关重要的映射人类糖组，通过挖掘创建的YSD库。 目的3-使用表征的VLRB的靶向诱变产生新的VLRB特异性，以产生定制的VLRB特异性。 具有潜在更高亲和力的抗体特异性。我们的研究的成功将与一个阿森纳的高潮 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 研究社区。这将最终鼓励对这些极其重要、复杂的 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， 允许研究人糖体的表达和功能。

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.