Rapid, Single-Step Monitoring of Monoclonal Antibody Bioavailability by Using a TNF-α-Based Multiepitope DNA Nanoswitch

Therapeutic drug monitoring (TDM) is increasingly valuable for tailoring personalized therapy, particularly in managing chronic inflammatory diseases where overtreatment can cause significant side effects. Monoclonal antibodies (mAbs), a primary therapeutic approach for these conditions, face challenges from antidrug antibodies (ADAs), which can reduce mAb bioavailability and efficacy. To address these issues, we utilized Tumor Necrosis Factor α (TNF-α) as a binding moiety in a fluorescence-based programmable nanosensor within the NanoHYBRID (NH) platform developed by Ulisse Biomed S.p.A. By directly conjugating TNF-α to DNA probes, we developed a rapid, homogeneous, affinity-based assay capable of detecting multiple mAbs targeting distinct epitopes on the same protein. This NH platform effectively detected therapeutic concentrations of clinically relevant mAbs, such as Infliximab, Adalimumab, and Golimumab, in blood serum samples in a one-step process, bypassing the need for time-intensive washing steps. Moreover, the NH sensor exhibited heightened sensitivity to the presence of ADA, which impacted drug quantification, indicating its utility for monitoring bioavailable mAb levels. Compared to ELISA, the NH platform demonstrated superior sensitivity to ADAs, suggesting its potential as a highly specific, modular solution for TDM. This modular design allows the NH platform to create multiepitope nanosensors capable of measuring bioavailable mAbs in a single step.