Abstract 1814: An integrated DMPK and bioanalytical platform for comprehensive characterization of antibody-drug conjugates (ADCs).

Abstract Introduction: Antibody-drug conjugates (ADCs) represent a transformative class of targeted oncology therapeutics; their complex structure introduces significant challenges in characterization, necessitating a holistic DMPK strategy to understand their in vitro stability, in vivo PK and biodistribution, and biotransformation. The success of this strategy hinges on the precise quantification of key analytes—including total antibody (Tab), conjugated antibody (ADC), free payload, and the drug-to-antibody ratio (DAR)—in diverse biological matrices. Here, we present an integrated DMPK platform evaluating Trastuzumab Deruxtecan (T-DXd), Enfortumab Vedotin (EV), and Trastuzumab Emtansine (T-DM1), with distinct linkers/payloads, to delineate PK behavior and biotransformation pathways. Methods: For three marketed ADCs, including T-DXd, EV, and T-DM1, in vitro plasma stability was assessed in human, monkey, rat, and mouse plasma (37 °C, 7-21 days) and followed a single 10 mg/kg i.v. dose for in vivo PK in naïve C57BL/6 mice. Quantitation used ligand binding assay (total and conjugated antibody), LC-MS/MS for free payload (LLOQ 10-50 pg/mL), and hybrid immunocapture LC-HRMS for DAR and biotransformation assessment. Results: T-DXd, with a cleavable peptide linker and high baseline DAR (∼7.6), demonstrated high stability in vitro, showing less than 40% DAR decrease (to ∼4.8) over 7 days and minimal free DXd release (2% across species). It’s in vivo DAR gradually declined to ∼4.0 by Day 14, with highly overlapped PK curves for Tab and conjugated ADC. EV, incorporating a protease-cleavable linker and a baseline DAR of ∼3.5, exhibited faster degradation in vitro, with DAR dropping to ∼1.6 and significant MMAE release (∼74 ng/mL at 24 h in mouse plasma). In vivo, ADC levels displayed a more rapid decline than Tab, accompanied by a marked DAR decrease to ∼0.5 by Day 21. T-DM1, featuring a non-cleavable thioether linker and DAR ∼3.5, displayed very high plasma stability and negligible free DM1 release (0.1 ng/mL). It’s in vivo PK profiles for Tab and ADC were nearly identical, confirming minimal payload release in circulation. For ADCs lacking anti-payload reagents, we also developed a hybrid approach combining DAR-insensitive Tab assay with LC-HRMS-based DAR profiling. This flexible bioanalytical strategy enabled comprehensive characterization across diverse ADC formats. Conclusion: Our integrated DMPK platform provides comprehensive and robust ADC characterization capability, promoting critical insights into ADC stability, biotransformation, and exposure profiles. This supports direct correlation between ADC design, in vitro properties, and in vivo PK behavior, thereby de-risking candidate selection and accelerating the development of novel ADC therapeutics. Citation Format: Kefeng Gong, Xinhe Feng, Zhengyi Hua, Yanting Ma, Rui Wang, Xiaolong Tu, Luke Yu. An integrated DMPK and bioanalytical platform for comprehensive characterization of antibody-drug conjugates (ADCs) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1814.