Development of a Mertansine-specific DNA Aptamer and Novel High-throughput Sandwich Enzyme-linked Oligonucleotide Assay for Quantification and Characterization of Trastuzumab Emtansine

We developed a novel DNA aptamer, D8#24S1, which specifically recognizes mertansine (DM1), the cytotoxic payload of the antibody-drug conjugate (ADC) trastuzumab emtansine (T-DM1), and applied it for T-DM1 analysis. D8#24S1 was obtained through SELEX and was shown to specifically recognize DM1 with high affinity (dissociation constant, K = 84.2 nM). By combining this anti-payload aptamer with the trastuzumab-specific anti-idiotype aptamer, CH1-S3, we developed a sandwich enzyme-linked oligonucleotide assay (sELONA) for evaluating T-DM1 content and drug-to-antibody ratio (DAR). The sELONA demonstrated an excellent fit to 4-parameter logistic curve model (R = 0.994) over a T-DM1 concentration range of 1-500 μg/mL, with a lower limit of quantification of 1 μg/mL, a precision within 23.9% (n = 3), and an accuracy within ±20.2% (n = 3). The sELONA also showed specificity when tested with other therapeutic monoclonal antibodies, such as trastuzumab and bevacizumab, as well as complex samples like serum. For DAR analysis, the sELONA exhibited high linearity (R = 0.988) and a strong correlation with hydrophobic interaction chromatography, a conventional method (R = 0.984). Unlike antibody-based assays, such as enzyme-linked immunosorbent assays, the sELONA employs chemically synthesized aptamers, offering superior robustness and cost-effectiveness. Additionally, when compared to conventional HIC, sELONA utilizes a 96-well microplate format, enabling high-throughput analysis. This study demonstrates the feasibility of aptamer-based assays as reliable alternatives to antibody-dependent methods, providing an efficient and adaptable approach for evaluating ADCs and potentially contributing to streamlined pharmaceutical development.