Abstract 7046: Payload and linker selection in novel osteosarcoma antibody-drug conjugate (ADC) targeting CADM1

Abstract Introduction: Our efforts in developing new therapies for osteosarcoma have focused on CADM1 as a novel target for ADC development. After successful proof of principle in vitro and in vivo tests, we developed three GMP manufactured CADM1-targeting ADCs with different linker and payload combinations: CADM1-PEG-Exatecan, CADM1-GGFG-Exatecan (a topoisomerase I inhibitor), and CADM1-Tesirine (a BPD-dimer). Materials and Methods: We tested 15 of our own osteosarcoma PDX-derived cell lines for cytotoxicity using AlamarBlue and Incucyte Sartorius assays. CADM1-Tesirine was tested at 500 nM and the other two ADCs at 1000 nM due to different cytotoxicity profiles, all three with a 10x dilution. The cells were incubated with the drugs for 120h. Internalization was assessed using Incucyte and Fabfluor Red Labeling Reagent at 2.5 µg/ml for all ADCs and controls, including an isotype control IgG1, bound to the respective linker-payload. The bystander effect was evaluated by incubating CADM1-negative cells with media derived from the 5-day incubation of both CADM1-positive and CADM1-negative cells. Results: CADM1-Tesirine showed significant cytotoxicity across all tested cell lines (IC50: 0.014 - 5.4 nM). CADM1-GGFG-Exatecan exhibited potent activity in most cell lines (IC50: 1.28 - 115 nM). CADM1-PEG-Exatecan had higher IC50 values (2.15 - 222 nM) but had better internalization than CADM1-GGFG-Exatecan. The fluorescence intensity increased over time, with a significant rise within the first 24 hours, which was not as consistently observed for CADM1-GGFG-Exatecan. CADM1-Tesirine displayed robust internalization across all cell lines, following a trend comparable to CADM1-PEG-Exatecan but often achieving even higher fluorescence intensity, thus proving to be the most internalized of the three ADCs. The bystander effect analysis showed that only the media derived from treated CADM1-positive cells induced cytotoxicity, as evidenced by the decreased growth rate using the Incucyte assay. Conclusions: All three ADCs demonstrate significant anti-tumor activity, however, CADM1-Tesirine had superior IC50 values and internalization. Results from the bystander experiments indicate target dependent internalization and linker cleavage. We are currently conducting in vivo tests to further evaluate their performance anticipating the conduct of a phase 1/2 trial in patients with recurrent osteosarcoma. Citation Format: Caterina Longo, Zhongting Zhang, Yifei Wang, Wendong Zhang, Zhaohui Xu, Yanhua Yi, Jonathan B. Gill, Micheal Roth, Xin Zhou, Richard Gorlick. Payload and linker selection in novel osteosarcoma antibody-drug conjugate (ADC) targeting CADM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7046.