Insights into erythropoietin homeostasis: Identifying the autoantigen targeted in the TEMPI syndrome

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Project Summary The TEMPI syndrome is a rare disorder characterized by five features: (1) Telangiectasias, (2) elevated Erythropoietin and erythrocytosis, (3) Monoclonal gammopathy, (4) Perinephric fluid collections and (5) Intrapulmonary shunting. This is a syndrome that we first identified and described in 2011 and have since found 27 patients worldwide. The TEMPI syndrome is an acquired disorder that appears to be driven by the presence of an abnormal autoantibody, i.e., the monoclonal gammopathy. In patients who have required treatment, therapies that target plasma cells and eradicate the antibody have led to a complete resolution of the other features of the TEMPI syndrome, implicating the antibody as a pathogenic driver of the disease. Patients with TEMPI syndrome present with serum erythropoietin (EPO) levels that can range as high as 500x the upper limit of normal. This suggests that the pathway of hypoxia sensing, and EPO production are dramatically dysregulated. Taken together, this further implicates the abnormal autoantibody as a pathogenic driver of EPO production. In this proposal we aim to identify the antigenic target of the monoclonal gammopathy. Understanding how this antibody-antigen interaction can drive both EPO production from the kidneys and drive neovascularization within the endothelium of the skin (telangiectasias) and lungs (shunting) has the potential to identify an entirely new signaling mechanism triggered by extracellular antibody binding. We will couple multiple approaches to antigen identification with gene expression and mutational profiling of the monoclonal plasma cells. Our approach takes advantage of available patient samples that have been carefully and longitudinally banked from patients of this ultra-rare disease. Our preliminary data demonstrates that we can successfully identify and clone the heavy and light chain monoclonal antibody sequences, and this has permitted the in vitro production of recombinant antibody from one patient already. In an exciting recent experiment, we have demonstrated that this recombinant antibody is capable of driving erythrocytosis when introduced into mice, lending strong support to our hypothesis that the monoclonal gammopathy is pathogenic. In this proposal, we will build on this preliminary data, and generate recombinant monoclonal antibodies from additional patients with the TEMPI syndrome will the goal of using these antibodies as tools for antigen identification. Pilot attempts using crude patient serum as a source of antibody for antigen identification have been unsuccessful, leading us to surmise that the antibody-antigen interaction may be of low-affinity. Thus, we are proposing a multi-pronged approach using both traditional immunology approaches as well as a new approach of proximity labeling. If successful, our proposal will identify the antigenic target of the monoclonal gammopathy in patients with TEMPI syndrome and in doing so will identify a novel antibody- antigen interaction that drives EPO production and neovascularization.