Evaluation of the Ligand‐Independent Activation of a VHH‐Based Anti‐CD19 SynNotch Receptor by Dual‐Luciferase Assay

Ligand-independent activation poses a significant challenge for synthetic receptors, limiting their dynamic range. Given the difficulties and inconsistencies in evaluating this crucial aspect in many studies, we utilized a dual-luciferase assay to assess the background activity of the SynNotch receptor. These receptors are modular, orthogonal constructs designed to detect and respond to specific extracellular signals by expressing a targeted protein. In this study, we engineered an anti-CD19 SynNotch receptor by incorporating a nanobody into the antigen-binding domain, leveraging the advantageous properties of camelid VHHs. We then evaluated the performance of the final construct, focusing on its functionality and ligand-independent activation using the dual-luciferase assay. The results revealed that although reporter expression significantly increased in the presence of the antigen, there was also a high level of background expression, which could hinder the performance of the SynNotch receptor. Additionally, the SynNotch receptor exhibited a 24-h delay in reaching its peak activation level in response to the antigen and in returning to baseline levels in its absence. Therefore, the optimization of the SynNotch construct is essential to enhance the receptor's dynamic range.