Abstract Background: ADC development in osteosarcoma has been hindered by the absence of effective, targetable surface antigens. We previously identified CADM1, a cell-surface adhesion molecule, as a promising therapeutic target and generated three CADM1-directed ADCs (CADM1-Tesirine, CADM1-PEG-Exatecan, and CADM1-GGFG-Exatecan) with distinct linker-payload combinations. To select the optimal candidate for clinical translation, we evaluated target engagement and target-dependent cytotoxicity across multiple in vitro models. Methods: Confocal microscopy was used to visualize ADC trafficking through cellular compartments and its lysosomal localization over time. CADM1 knockout (KO) osteosarcoma cell lines (SaOS2 and OS31) were generated using CRISPR/Cas9 and validated by flow cytometry, Western blotting, and mass spectrometry (MS). Cell-surface CADM1 levels were quantified by MS and correlated with ADC cytotoxicity (IC50 values) measured by real-time live-cell imaging on the IncuCyte platform. Spearman correlation analyses across nine osteosarcoma cell lines assessed the relationship between CADM1 expression and ADC potency for all three constructs. Results: Confocal time-course imaging showed ADC trafficking from the plasma membrane (30 min) to lysosomes (6 h) and cytoplasmic dispersion (24 h), absent in CADM1-KO cells, confirming target-mediated internalization and lysosomal processing. CRISPR/Cas9-mediated knockout of CADM1 was achieved in SaOS2 and OS31 osteosarcoma cell lines. Loss of CADM1 expression was confirmed by flow cytometry, Western blotting, and mass spectrometry, showing complete depletion of surface protein. CADM1 loss markedly reduced ADC cytotoxicity (IC50 = 77.41 nM in SaOS2 CADM1-KO vs 0.014 nM in SaOS2), confirming on-target activity. Across nine osteosarcoma models, CADM1 surface abundance strongly correlated with drug potency, most notably for CADM1-GGFG-Exatecan (Spearman ρ = -0.98, p = 0.001). These data identify CADM1 as a determinant of ADC efficacy and highlight the contribution of other ADC components. Conclusions: These data demonstrate potent, target-dependent activity of the generated CADM1-targeted ADCs, validating CADM1 as a rational therapeutic target. Among them, CADM1-GGFG-Exatecan showed the strongest correlation between IC50 and target expression, supporting selective cytotoxic delivery. In vivo efficacy and toxicology studies are ongoing to finalize candidate selection. Citation Format: Caterina Longo, Zhongting Zhang, Wendong Zhang, Yifei Wang, Adil Bahadir, Yi Yanhua, Zhaohui Xu, Xin Zhou, Michael Roth, Jonathan Gill, Richard Gorlick. Surface CADM1 (Cell Adhesion Molecule 1) abundance predicts payload delivery and in-vitro efficacy of CADM1-GGFG-Exatecan ADC in osteosarcoma, aiding optimal candidate selection 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 7883.
Longo et al. (Fri,) studied this question.