A scalable pipeline for generating synthetic antibodies against designer glycotopes

1605242 DeLisa，Matthew P.碳水化合物链，称为聚糖，参与细胞的许多功能，因为它们决定了大分子如蛋白质的性质。 不幸的是，很难确定聚糖的类型和结构。 然而，识别并结合特定聚糖的抗体可以为这些碳水化合物的分析提供强大的工具。 该提案的目的是创建一种稳健的方法来生成聚糖识别抗体，该抗体将结合特定链并可用于分析这些结构。 更好地了解聚糖的作用将有助于了解细胞行为的基础知识，这对于寻找疾病的治疗方法和在生物制造中工程化高效细胞至关重要。该提案的目标是创建一个强大的集成管道，用于快速发现和表征选择性，针对具有生物医学重要性的确定聚糖结构或聚糖-多肽决定簇的高亲和力Ab。潜在的假设是，糖工程化的大肠杆菌可以被利用来容易地生产大量的相对纯的糖表位，其可以被有效地固定到固体支持物上，并用于从噬菌体展示的合成Ab文库中平行选择糖体。为了验证这一假设，将使用携带糖基转移酶合成途径的工程化细菌合成约100种结构均匀的糖表位。然后，使用准手动方案，通过Fab噬菌体展示的迭代轮次从大型组合文库中选择针对这些糖表位的合成糖体。最后，将使用ELISA、表面等离子体共振（SPR）和聚糖微阵列表征分离的糖体的糖表位亲和力和特异性。为了建立生物学相关性，糖体将用于探测原代细胞系、病毒或病原性细菌上真实聚糖特征的表达。通过在同一个实验室内将糖抗原表达（Aim 1）与抗体选择（Aim 2）结合起来，该项目预计将在几周内产生一个用于常规生成正交糖体-糖表位对的集成管道。CBET部门生物技术和生物化学工程项目的这一奖项由材料研究部门生物材料项目共同赞助。

1605242 DeLisa, Matthew P.Carbohydrate chains, known as glycans, are involved in many functions of a cell since they determine the properties of macromolecules such as proteins. Unfortunately it is difficult to determine the type and structure of glycans. However, antibodies that recognize and bind to specific glycans could offer a powerful tool for the analysis of these carbohydrates. The objective of this proposal is to create a robust method to generate glycan recognizing antibodies that will bind to specific chains and that can be used to analyze these structures. A better understanding of the role that glycans play will contribute to the fundamental knowledge of the behavior of cells which is critical for finding cures for diseases and for engineering efficient cells in biomanufacturing.The objective of this proposal is to create a robust, integrated pipeline for the rapid discovery and characterization of selective, high-affinity Abs against defined glycan structures or glycan-polypeptide determinants of biomedical importance. The underlying hypothesis is that glycoengineered Escherichia coli can be leveraged for facile production of large quantities of relatively pure glycotopes, which can be efficiently immobilized to solid supports and used for parallel selection of glycobodies from phage-displayed synthetic Ab libraries. To test this hypothesis, an array of ~100 structurally uniform glycotopes will be synthesized using engineered bacteria carrying synthetic pathways of glycosyltransferases. Then, using a quasi-manual protocol, synthetic glycobodies against these glycotopes will be selected from large combinatorial libraries by iterative rounds of Fab-phage display. Finally, isolated glycobodies will be characterized for glycotope affinity and specificity using ELISA, surface plasmon resonance (SPR), and glycan microarrays. To establish biological relevance, glycobodies will be used to probe the expression of authentic glycan signatures on primary cell lines, viruses, or pathogenic bacteria. By coupling glyco-antigen expression (Aim 1) to antibody selection (Aim 2) within the same laboratory, this project is anticipated to yield an integrated pipeline for routine generation of orthogonal glycobody-glycotope pairs in a matter of weeks.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is cosponsored by the Biomaterials Program of the Division of Materials Research.