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

摘要 这项申请解决了一个主要的未得到满足的技术需求，这将促进糖组分和 糖链表达与基因组学、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和动物的多糖在 在正常发育、动态平衡和疾病过程的背景下。糖科学中的传统研究 依赖于一种奇怪的植物来源的凝集素和一些传统的鼠类单抗， 但这类试剂有多个局限性。这个应用程序利用了我们的突破性技术，使我们能够 开发一套特异的、具有严格特征的抗碳水化合物抗体(ACAs)。我们已经开发出 使用可变淋巴细胞受体(VLRBs)的高通量免疫和筛选技术 用细胞、细胞结合物、糖蛋白或组织免疫古代七鳃鳗而产生的。 因为与哺乳动物相比，它们的免疫系统和糖类的进化多样性， 这些古老的鱼类产生了各种具有精致特异性的抗碳水化合物VLRBs，它们是 能够区分不同的连接，N-和O-聚糖上的基序呈现，以及是否存在 单糖上的官能团。免疫后，克隆的编码VLRBs的cDNA 在酵母表面展示(YSD)文库中表达，该文库包含多种个体特异性。这是一项创新 技术发展使我们既可以在单一免疫方案中生成VLRb库，也可以 以及使用我们众多和多样化的多糖和 糖肽微阵列和显示技术。最终，在对VLRb进行筛选以确定其与 表达了糖链抗原、重组和可复制的VLRB-Ig嵌合体，该嵌合体可用于所有类型 探索多糖的表达和功能的免疫学方法。我们提出了三个稳健和创新的建议 解决当前技术缺陷的具体目标：目标1--改进和扩展我们的技术，以 针对不同的人多糖靶向生成永久性的VLRB YSD文库，使用多个 免疫和筛查战略。目标2-丰富和鉴定特异性的单克隆性VLRBs 对于人类糖链，这将是绘制人类糖链的关键，通过挖掘创建的YSD文库。 目标3-利用特化的VLRb的定向突变产生新的VLRB特异性，以创建定制的VLRB 具有潜在较高亲和力的抗体特异性。我们研究的成功将伴随着一大批 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。这最终将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，以及 允许研究人类糖蛋白的表达和功能。

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.