Serum proteomics reveals distinct phenotypic signatures to IL-6 blockade between two immunotherapies

A recent clinical study tested the effects of two different monoclonal antibodies (mAbs) (siltuximab, anti-IL6; tocilizumab, anti-IL6R) on the fate and function of T-cells in people with type 1 diabetes. While both mAbs affect the response of T-cells to stimulation, they have very different, sometimes opposing mechanisms. Here, we use mass-spectrometry based proteomics to analyze longitudinal serum samples (baseline and two weeks post-treatment) from 20 clinical trial participants to examine the effects of siltuximab and tocilizumab on extracellular vesicles. To accomplish this, serum samples were enriched for extracellular vesicles with Mag-Net and analyzed by LC-MS/MS to identify significantly differentially abundant protein groups and pathways. Proteome analysis confirmed highly reproducible measurements across multiple draw dates. In total, we quantified >3300 protein groups of which 46 protein groups had significantly altered abundance after mAb treatment. Tocilizumab altered pathways associated with proteostasis (neddylation) and pre-notch transcription and translation. Siltuximab altered FCGR activation pathway members. In addition, quantitation of the monoclonal antibody therapies themselves enabled the measurement of the correlation between drug amounts and impacted proteins. Taken together, this work demonstrates the utility of the Mag-Net method to evaluate the impacts of therapeutic interventions on serum extracellular vesicles.