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

Background

Cytokines 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.


Objectives

Identify antibodies and antibody fragments that interact with TPOR and EPOR

Characterise their functionality as surrogate cytokines; monitoring receptor activation, downstream signalling and haematopoiesis.


Novelty

The 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.


Timeliness

We 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 approach

Antibody 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.