Designed Peptides as Affinity Ligands for Extracellular-Vesicle-based Cancer Biomarker Detection

Assays for cancer diagnosis via the analysis of tumor biomarkers on circulating extracellular vesicles (EVs) have shown great potential. Single EV imaging that can measure the abundance of protein biomarkers in EVs can help in detecting the presence, stage, and progression of disease. Antibodies are typically used to detect EV proteins. Controlling the quality of antibody-based immunoassays can, however, be challenging, as they may exhibit unintended cross-reactivity with non-target proteins or variability in binding affinity across different batches, even for monoclonal antibodies. Here, we report short peptides that are a promising alternative to antibodies for detecting protein biomarkers in EVs. We describe an effort that combines a Peptide Binding Design (PepBD) algorithm and molecular-level simulations to identify peptides that can recognize (1) the extracellular domain of EpCAM (a known cancer biomarker) and (2) the extracellular domain of tetraspanin CD81(a protein commonly expressed on the surface of EVs). The peptides designed for EpCAM and CD81 were labeled with a fluorescent dye and their binding to the target protein was evaluated using fluorescence ELISA. The results demonstrated that one of the computationally designed peptides EP-2.1 exhibited an affinity for the target EpCAM protein comparable to that of an antibody. Further testing involved single EV imaging to gauge the peptides affinity towards EVs. Designed peptides for both the target proteins showed similar affinity as antibodies to EVs.