Fine-tuning blood cell development: Using antibodies as surrogate cytokines to modify receptor activation and signalling in haematopoiesis

微调血细胞发育：使用抗体作为替代细胞因子来改变造血过程中的受体激活和信号传导

• 批准号: 2752707
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2752707
• 关键词: Fine; tuning; blood; cell; development

BackgroundCytokines are a large family of functionally diverse glycoproteins that are fundamental regulators of haematopoiesis, controlling blood cell development, haematopoietic stem cell (HSC) maintenance and immunity. Remarkably, just two cytokines, erythropoietin (EPO) and thrombopoietin (TPO), are almost entirely responsible for the production of >99% of circulating blood cells, with millions of erythrocytes and platelets generated every second. Consequently, aberrant EPO and TPO signalling can have profound pathological effects on haematopoiesis, ranging from aplastic anaemia to haematological malignancies. By understanding how we can use biological agents that bind to and alter EPO and TPO receptor function, we can potentially develop surrogate cytokines that are able to moderate receptor activation, downstream signalling and blood cell development. ObjectivesIdentify antibodies and antibody fragments that interact with TPOR and EPOR Characterise their functionality as surrogate cytokines; monitoring receptor activation, downstream signalling and haematopoiesis. NoveltyThe potential of surrogate cytokine molecules to control receptor activity with greater precision has only been realised over the last few years. This project extends the investigations to include two of the most essential cytokine receptors that impact haematopoiesis at multiple stages. TimelinessWe have recently uncovered a new mechanistic paradigm in cytokine signalling whereby TPO and EPO receptors exist at the cell surface as monomers, rather than as pre-formed dimers, and dimerize in response to their cognate ligands (Science, 2020). Not only did these findings highlight the significant complexity of receptor activation, it now provides us with the opportunity to manipulate these receptors and precisely control signalling outcomes. Experimental approachAntibody and antibody fragments binding to TPOR and EPOR will be generated in vitro by phage display. A broad panel of antibodies that show diverse binding characteristics and epitopes will be identified by biochemical and biophysical high throughput screening. Functional characterization of antibody and antibody fragments will involve cellular assay to determine effects on growth and differentiation, barcoded phospho-flow cytometry to understand differences in signalling outputs and single molecule total internal fluorescence (smTIRF) microscopy to quantify changes in cognate ligand-induced and ligand-independent dimerization. Selected molecules will progress to further analysis including primary cell assays to determine effects on HSC maintenance and lineage differentiation in vitro and in vivo characterisation using mice to identify impacts on erythrocyte and megakaryocyte differentiation and HSC maintenance.

背景循环因子是一个功能多样的糖蛋白，是造血的基本调节剂，控制血细胞发育，造血干细胞（HSC）维持和免疫力。值得注意的是，只有两种细胞因子，促红细胞生成素（EPO）和血小板素（TPO）几乎完全负责产生> 99％的循环血细胞，每秒产生数百万个红细胞和血小板。因此，异常的EPO和TPO信号传导可能会对造血症产生深远的病理影响，从性质贫血到血液学恶性肿瘤。通过了解如何使用结合并改变EPO和TPO受体功能的生物学剂，我们可以发展能够中度受体激活，下游信号传导和血细胞发育的替代细胞因子。目标识别与TPOR相互作用并EPOR相互作用的抗体和抗体片段的功能将其功能描述为替代细胞因子。监测受体激活，下游信号传导和造血。新颖的替代细胞因子分子对以更精确的方式控制受体活性的潜力仅在过去几年中才实现。该项目将研究扩展到包括在多个阶段影响造血的两个最重要的细胞因子受体。 Timeliness我们最近在细胞因子信号传导中发现了一种新的机械范式，在细胞因子信号传导中，TPO和EPO受体在细胞表面以单体而不是预先形成的二聚体存在，并响应其同源配体（Science，2020年）。这些发现不仅突出了受体激活的显着复杂性，现在还为我们提供了操纵这些受体并精确控制信号传导结果的机会。实验性方法抗体和与TPOR结合的抗体片段将通过噬菌体显示在体外产生。一系列显示出多种结合特征和表位的抗体将通过生化和生物物理高吞吐量筛选来鉴定。抗体和抗体片段的功能表征将涉及细胞测定，以确定对生长和分化的影响，条形码磷酸化的磷酸化细胞仪，以了解信号输出和单分子总内荧光（SMTIRF）显微镜的差异，以量化认知配体诱导的诱导的和ligand诱导的和ligand诱导的变化。选定的分子将进行进一步分析，包括主要细胞测定法，以确定对HSC维持的影响，并使用小鼠在体外和体内表征分化的影响，以识别对红细胞和巨核细胞分化和HSC维持的影响。