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庭，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，红细胞生成素（EPO）和血小板生成素（TPO）这两种细胞因子几乎完全负责99%的循环血细胞的产生，每秒产生数百万个红细胞和血小板。因此，异常的EPO和TPO信号可以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系统恶性肿瘤。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激活、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。在过去的几年里，替代细胞因子分子以更高的精度控制受体活性的潜力才得以实现。该项目将研究扩展到包括两个最重要的细胞因子受体，它们在多个阶段影响造血。我们最近发现了细胞因子信号传导的一种新的机制范式，即TPO和EPO受体以单体形式存在于细胞表面，而不是以预先形成的二聚体形式存在，并根据其同源配体进行二聚（Science, 2020）。这些发现不仅突出了受体激活的显著复杂性，它现在为我们提供了操纵这些受体和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。通过生物化学和生物物理高通量筛选，将鉴定出具有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包括细胞分析，以确定对生长和分化的影响，条形码磷酸化流式细胞术，以了解信号输出的差异，单分子总内荧光（smTIRF）显微镜，以量化同源配体诱导和不依赖配体的二聚化的变化。选定的分子将进行进一步的分析，包括原代细胞分析，以确定对HSC维持和体外谱系分化的影响，以及使用小鼠进行体内表征，以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.