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Abstract During the last decade antibody-drug-conjugates (ADCs) have become an important and validated treatment modality for cancer patients. Here, we introduce a unique next generation ADC platform based on Sortase-mediated antibody conjugation (SMAC-Technology) yielding in very homogenous and stable drug conjugates with limited systemic, but powerful anti-tumor activity. The highly potent payload used is a proprietary derivative of the Anthracycline PNU-159682. Our PNU-159682-based ADCs are known to not only induce DNA damage in the target cell, but importantly also trigger immunogenic cell death and thus, stimulating anti-tumor immunity offering untapped combination potential. ADCs based on the SMAC-Technology platform currently undergo clinical development. In this work, we present functional studies that were performed to further elucidate the mode of action (MoA) of the ADC payload PNU-159682 and to investigate any potential liabilities with respect to sensitivity, which could limit treatment options for cancer patients. Flow cytometry-based readouts verified that PNU-EDA, a derivative of PNU-159682, is a highly potent inducer of DNA damage, cell cycle arrest and cell death. Interestingly, PNU-EDA arrests cells and inhibits DNA synthesis in S-phase in contrast to other Anthracyclines such as Doxorubicin, which leads to a block in the G2/M-phase. High-throughput cell line panel sensitivity screens confirmed that the activity of PNU-EDA is broad and not restricted to specific cancer indications. Notably, a correlation between lower potency and increased ABCB1 (MDR1) expression levels was observed for PNU-EDA, but not for parental PNU-159682. More detailed mechanistic insights were obtained by performing a genome-wide CRISPR-Cas9 KO screen, which revealed a partial dependency of the PNU-EDA potency on the proficiency of the TC-NER DNA damage repair mechanism. This finding further highlights a distinct MoA of PNU-159682 compared to Doxorubicin or other DNA-targeting compounds such as Cisplatin. Overall, our screens and mechanistic studies verified that PNU-159682-based ADCs efficiently kill cancer cells via a particular MoA and thereby, offer highly promising therapeutic options to a diverse and large population of cancer patients. Citation Format: Monika Kaiser, Ulrike Fiedler, Daniel Gerlach, Norbert Kraut, Lukas Bammert. The ADC payload PNU-159682 is highly active in a wide range of cancer indications by inducing DNA damage and cell death via a distinct mode of action abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3143.
Kaiser et al. (Fri,) studied this question.