Abstract The development of effective anti-cancer drugs relies on accurately evaluating therapeutic antibodies. Three-dimensional (3D) tumoroids, which closely mimic the tumor microenvironment, help provide a promising platform for these evaluations. Our research focuses on validating therapeutics using antibody degradation trackers and antibody drug conjugates within 3D tumoroids. LysoLight tagged antibodies are fluorogenic in lysosomes, allowing visualization of antibody behavior within tumoroids, enabling insights into antibody degradation. Our studies showed higher Cetuximab uptake and localization to lysosomes in EGFR-overexpressing lung adenocarcinoma tumoroids, while non-specific antibodies or EGFR-null tumoroids showed minimal LysoLight activity. Additionally, we generated MMAE conjugated therapeutic antibodies using the new SiteClick rapid antibody conjugation technique, that allowed rapid screening of candidates from selected monoclonal antibodies to identify candidates with favorable therapeutic potential. High-throughput assays within 3D tumoroids enabled us to evaluate binding affinity, internalization rates, and cytotoxic effects. Our findings indicated that a MMAE conjugated EGFR targeted antibody-drug conjugate, facilitated a targeted cell penetration of drugs in colon cancer tumoroids and induced cell death. Integrating antibody degradation trackers and high-throughput Ab screenings within 3D tumoroids offers a robust platform for evaluating therapeutic efficacy, by providing a more accurate representation of their behavior in vivo. Disclaimer: For Research Use Only. Not for use in diagnostic procedures. © 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Citation Format: Jayanth Surya Narayanan Shankara Narayanan, Ryan Holly. Next generation molecular tools for evaluating antibody drug conjugates in 3D systems 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 4447.
Narayanan et al. (Fri,) studied this question.