A sensitive MSD-ECL platform for in vivo pharmacokinetic profiling of ADCs

Antibody-drug conjugates (ADCs) are a rapidly evolving class of targeted cancer therapeutics that combine the specificity of monoclonal antibodies with the potency of small-molecule cytotoxic drugs. It is critical for ADCs to be stable in the systemic circulation in order to prevent premature release of the payload, which can lead to off-target toxicity and reduced therapeutic efficacy. The pharmacokinetic (PK) behavior of ADCs is influenced by several factors, such as antibody structural heterogeneity, linker type, and payload physicochemical properties. Stability is a key quality attribute that directly affects dosing strategies, the therapeutic window, and clinical outcomes. Therefore, robust and sensitive analytical platforms are essential for monitoring ADC integrity and payload release over time. While traditional ELISA-based methods are widely used, they often lack the sensitivity and dynamic range required to detect subtle changes in ADC composition during circulation. In this study, we use the Meso Scale Discovery® (MSD) platform, which uses plate-based electrochemiluminescence (ECL) technology, to evaluate the in vivo PK and stability of ADCs. By applying this technology to preclinical in vivo models, we aim to generate high-resolution PK profiles and stability data that support the rational design and optimization of ADCs for clinical development. Standard 1-Spot SECTOR plates were used to detect the antibody backbone by coating them with a specific capture antibody. Small Spot Streptavidin SECTOR plates were used to detect the intact ADC, capturing the molecule via anti-payload antibodies. In both assays, detection was performed using a sulfo-tagged anti-human IgG antibody. After optimizing the setup, it demonstrated a broad dynamic range of 4-5 orders of magnitude and a lower limit of detection of less than 100 pg/mL for both the antibody and the intact ADC. Subsequent in vivo PK studies involved administering ADCs via a single intravenous injection to SCID beige mice. Serum samples were diluted 1:1000 in PBS containing 1% BSA, and 25 µL per well was applied to the MSD plates. This high dilution factor permits minimal blood volume collection per time point, thereby reducing the number of animals required. The assay demonstrated excellent reproducibility with minimal variability between replicates. To further increase the relevance of the results, future studies may use mFcRn⁻/⁻ hFcRn transgenic mice or HSA/hFcRn/hFcgammaR triple transgenic models. These models more accurately reflect the serum half-life of human IgG and correlate well with data from cynomolgus monkeys and humans. In summary, this MSD-based assay platform offers a highly sensitive and reproducible approach for in vivo PK analysis of ADCs that spares animals. There is potential to further refine this approach using humanized mouse models to improve clinical predictability.