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

细胞因子是一个功能多样的糖蛋白大家族，是造血的基本调节因子，控制血细胞发育、造血干细胞（HSC）维持和免疫。值得注意的是，只有两种细胞因子，促红细胞生成素（EPO）和促血小板生成素（TPO），几乎完全负责生产>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信号传导可对造血产生深远的病理影响，从再生障碍性贫血到血液恶性肿瘤。通过了解我们如何使用结合并改变EPO和TPO受体功能的生物制剂，我们可以潜在地开发能够调节受体活化、下游信号传导和血细胞发育的替代细胞因子。目的鉴定与TPOR和EPOR相互作用的抗体和抗体片段，表征其作为替代细胞因子的功能;监测受体活化、下游信号传导和造血。新颖性替代细胞因子分子以更高精度控制受体活性的潜力仅在过去几年中才得以实现。该项目将调查扩展到包括两种在多个阶段影响造血的最重要的细胞因子受体。我们最近发现了细胞因子信号传导中的一种新的机制范式，其中TPO和EPO受体作为单体而不是作为预形成的二聚体存在于细胞表面，并响应于其同源配体而二聚化（Science，2020）。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结果的机会。实验方法利用噬菌体展示技术在体外制备与TPOR和EPOR结合的抗体和抗体片段。将通过生物化学和生物物理高通量筛选鉴定显示不同结合特征和表位的一组广泛的抗体。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）显微镜以定量同源配体诱导和配体非依赖性二聚化的变化。所选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，并使用小鼠进行体内和体外表征，以确定对红细胞和巨核细胞分化以及HSC维持的影响。

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.