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.

背景细胞因子是一个功能多样的糖蛋白大家族，是造血、控制血细胞发育、造血干细胞（HSC）维持和免疫的基本调节因子。值得注意的是，只有两种细胞因子，即促红细胞生成素 (EPO) 和血小板生成素 (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 信号传导会对造血产生深远的病理影响，范围从再生障碍性贫血到血液恶性肿瘤。通过了解如何使用结合并改变 EPO 和 TPO 受体功能的生物制剂，我们有可能开发出能够调节受体激活、下游信号传导和血细胞发育的替代细胞因子。目标鉴定与 TPOR 和 EPOR 相互作用的抗体和抗体片段，表征它们作为替代细胞因子的功能；监测受体激活、下游信号传导和造血作用。新颖性 替代细胞因子分子更精确地控制受体活性的潜力直到最近几年才被认识到。该项目将研究范围扩大到包括在多个阶段影响造血的两种最重要的细胞因子受体。及时性我们最近发现了细胞因子信号转导的新机制范例，其中 TPO 和 EPO 受体作为单体而不是预先形成的二聚体存在于细胞表面，并响应其同源配体而二聚化（Science，2020）。这些发现不仅强调了受体激活的显着复杂性，而且现在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。实验方法通过噬菌体展示在体外产生与TPOR和EPOR结合的抗体和抗体片段。将通过生化和生物物理高通量筛选来鉴定表现出不同结合特征和表位的广泛抗体。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，条形码磷酸流式细胞术，以了解信号输出的差异，以及单分子总内部荧光（smTIRF）显微镜，以量化同源配体诱导和配体独立二聚化的变化。选定的分子将进行进一步分析，包括原代细胞测定，以确定对 HSC 维持和谱系分化的影响，并使用小鼠进行体外和体内表征，以确定对红细胞和巨核细胞分化以及 HSC 维持的影响。