Engineer Immune Cells via Chemoenzymatic Glycan Editing

项目摘要/摘要 细胞间的相互作用涉及许多生物过程，包括免疫反应， 胚胎发育、神经信号与癌症转移。我们在认识上的一些重大差距 细胞-细胞相互作用包括：在不同阶段，特定细胞类型的相互作用伙伴是什么 生物过程？如何修改这些交互伙伴的细胞表面景观以调整功能 一种内源性功能扰动最小的特定细胞类型？ 本项目的目标是开发基于糖基转移酶的化学酶方法来 在细胞表面加入生物聚合物，包括酶和单抗，以研究这些 拼图。 在这个项目的目标1中，我们将确定或设计一种可以转移的混杂糖基转移酶 生物大分子功能化的非天然糖从相应的核苷酸供体到细胞表面 感兴趣细胞的糖萼。当生物多聚体是一种酶时，它将使邻近依赖 标记与含有酶的目标细胞(诱饵细胞)相互作用的猎物细胞，前提是 被捕食细胞的细胞表面表达被酶修饰的底物。随后，该信号 通过标记产生的产物可以通过流式细胞仪进行体外检测和分析。在目标1和目标2中，我们 将应用这种方法通过小鼠模型探测体内细胞与细胞的相互作用。重点将放在 免疫激活过程中特定免疫细胞类型与T细胞相互作用伙伴的鉴定 衰竭(T细胞衰竭是指T细胞逐渐丧失功能的过程)。最后，我们 将在目标3中开发一种方法，将单抗偶联到免疫细胞上，赋予它们 特定的目标能力。经修饰的细胞的迁移和抗肿瘤效果将在 小鼠肿瘤模型。

PROJECT SUMMARY/ABSTRACT Cell-cell interactions are involved in numerous biological processes, including immune responses, embryonic development, neuronal signaling and cancer metastasis. Some major gaps in our understanding of cell-cell interactions include: what are the interacting partners of a specific cell type at different stages of a biological process? How to modify the cell-surface landscape of these interacting partners to tune the functions of a specific cell type with minimum perturbation of its endogenous functions? The objective of this project is to develop glycosyltransferase-based chemoenzymatic methods to incorporate biopolymers, including enzymes and monoclonal antibodies, onto the cell surface to study these puzzles. In Aim 1 of this project, we will identify or engineer a promiscuous glycosyltransferase that can transfer biopolymer-functionalized unnatural sugars from the corresponding nucleotide sugar donors to the cell-surface glycocalyx of the cells of interest. When the bioploymer is an enzyme, it will enable the proximity-dependent labeling of prey cells that interact with the cells of interest (bait cells) that harbor the enzyme, providing that the cell surface of prey cells expresses the substrates to be modified by the enzyme. Subsequently, the signal generated by the labeling can be detected and analyzed ex vivo by flow cytometry. In Aim 1d and Aim 2, we will apply this method to probe cell-cell interactions in vivo using mouse models. The focus will be the identification of interacting partners of specific immune cell types during immune activation and T-cell exhaustion (T-cell exhaustion is the process during which T cells progressively lose their functions). Finally, we will develop a method in Aim 3 to conjugate monoclonal antibodies onto immune cells to endow them with specific targeting capabilities. The migration and anti-tumor efficacy of the modified cells will be evaluated in mouse tumor models.