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、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，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，每秒钟产生数百万红细胞和血小板。因此，EPO和TPO信号的异常可以在从再生障碍性贫血到血液系统恶性肿瘤的造血过程中产生深远的病理效应。通过了解我们如何使用生物制剂来结合和改变EPO和TPO受体的功能，我们可以潜在地开发能够调节受体激活、下游信号传递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，表征它们作为替代细胞因子的功能；监测受体激活、下游信号和造血。新奇的是，替代细胞因子分子更精确地控制受体活性的潜力只是在过去几年才实现的。该项目扩大了研究范围，包括在多个阶段影响造血的两种最基本的细胞因子受体。时间我们最近在细胞因子信号传递中发现了一种新的机制范式，根据该范式，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，并根据其同源配体而发生二聚体(Science，2020)。这些发现不仅突显了受体激活的显著复杂性，而且现在为我们提供了操纵这些受体并精确控制信号结果的机会。实验方法利用噬菌体展示技术在体外产生与TpoR和EpoR结合的抗体和抗体片段。通过生化和生物物理的高通量筛选，将鉴定出一系列表现出不同结合特征和表位的抗体。抗体和抗体片段的功能表征将涉及细胞分析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的和非配体非依赖性二聚体的变化。选定的分子将进行进一步的分析，包括原代细胞分析，以确定在体外和体内对HSC维持和谱系分化的影响，并使用小鼠来确定对红细胞和巨核细胞分化和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.