Spatially Driven DNA-Based Probe Proximity Assay for Total Antibody Quantification in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) face challenges in accurate total antibody quantification due to their complex composition, low in vivo concentrations, and interference from the serum matrix. The dynamic decrease in the drug-antibody ratio (DAR) during metabolism, varying affinities among different DAR species, and discrepancies between standards and actual samples further compromise the analytical accuracy. Herein, inspired by an aptamer targeting the non-complementarity determining region (nCDR), we developed a novel spatially driven DNA-based probe proximity assay for rapid, sensitive, and high-throughput total antibody quantification, using two oligonucleotides separately labeled with fluorescent or quenching groups. The high affinity and selectivity of the probes for trastuzumab and ADCs were confirmed by microscale thermophoresis, native PAGE, and molecular docking. Systematic spatial screening was used to further identify the optimal stem, spacer, and orientation between the two probes for collaborative recognition. Moreover, the proposed homogeneous detection method achieved superior recovery for trastuzumab and successfully quantified total antibodies in trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In contrast to the conventional indirect enzyme-linked immunosorbent assay (ELISA), our method reduces the recovery loss caused by decreased affinity from payload conjugation and delivers comparable signals for trastuzumab, T-DM1, and T-DXd, which provides a novel approach to address accuracy issues arising from affinity changes due to ADC dynamics in vivo. Finally, the proposed assay was applied to a series of spiked serum samples and clinical samples, demonstrating its feasibility and proof of concept. This strategy may facilitate the development of bioanalytical techniques for ADC characterization and monitoring.