Abstract 6392: Next-generation ADCs: precise delivery of exatecan to tumor nuclei by targeting the tumor microenvironment with an anti-DNA cell-penetrating antibody

Abstract Designing targeted therapies that selectively eliminate tumor cells while sparing healthy tissues is a major goal in oncology, where antibody-drug conjugates (ADCs) have emerged as promising tools. Traditional ADCs rely on specific antigens on tumor cell surfaces, limiting their applicability to cancers that overexpress them. This study introduces an innovative approach redefining ADC technology, bypassing the need for cell surface antigen recognition and utilizing a novel DNA-targeting mechanism. We present V66, a lupus-derived, cell-penetrating, anti-DNA antibody engineered to deliver Exatecan, a potent Topoisomerase I inhibitor, directly to tumors. V66 offers a breakthrough approach by recognizing and binding to extracellular DNA (ecDNA) within the tumor microenvironment, which accumulates due to high rates of cell turnover, death and necrosis, providing a unique target absent in normal tissues. Upon binding to ecDNA, V66 enters tumor cells via the Equilibrative Nucleoside Transporter (ENT2), overexpressed in various cancers but minimal in healthy cells, broadening ADC applicability. Interestingly, V66 accumulates to a greater extent in DDR-deficient cell lines in vitro, with this difference disappearing when ecDNA is removed, suggesting that elevated levels of ecDNA enhance V66 penetration. Fluorescently labeled V66-Exatecan confirmed its selective accumulation in tumors in vivo, with minimal signal to healthy tissues, underscoring its specificity and low toxicity potential. In vitro and in vivo experiments showed that V66-Exatecan localizes to the nucleus, where Exatecan inhibits Topo-I, activating the DNA Damage Repair pathway and triggering cell death. This approach demonstrated potent anticancer activity, with low nanomolar EC50 values across diverse cancer cell lines. Preclinical models, including cell line-derived xenografts (CDX) and genetically engineered mouse models (GEMMs), showed significant tumor regression and improved survival in hard-to-treat cancers such as triple-negative breast cancer (TNBC) and medulloblastoma, without the systemic toxicity typical of Topo-I inhibitors. V66-Exatecan also showed enhanced efficacy in DDR-deficient cells, which are particularly vulnerable due to impaired DNA repair mechanisms. Remarkably, we observed a significant percentage of complete regression in treated DDR-deficient tumors. Extensive toxicology studies established a substantial safety margin, with toxicity observed only at extremely high doses. The dual-targeting mechanism, which leverages both ecDNA presence and ENT2-mediated entry, sets V66-Exatecan apart from conventional ADCs, offering a versatile and safe option across diverse tumor types. This study lays the groundwork for further clinical exploration, representing a significant step toward adaptable, effective cancer therapies. Citation Format: Zaira Ianniello, Elias Quijano, Huimei Lu, Denise Hegan, Venu Bommireddy, Dale L. Ludwig, Zhiyuan Shen, Peter M. Glazer. Next-generation ADCs: precise delivery of exatecan to tumor nuclei by targeting the tumor microenvironment with an anti-DNA cell-penetrating antibody [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 6392.