Harnessing ExDNA for precision exatecan delivery in cancer: a novel antibody-drug conjugate approach

Background: Current antibody-drug conjugates (ADCs) face limitations due to a lack of tumor-selective targets, inefficient internalization, and challenges in reaching tumors in challenging sites, ultimately limiting their therapeutic efficacy. We developed and characterized V66-exatecan, a novel ADC composed of V66, a humanized antibody with high affinity for extracellular DNA (exDNA), conjugated to exatecan via a Cleavable Linker. This ADC employs a dual-targeting mechanism based on exDNA and ENT2 transporter expression to enhance nuclear drug delivery and tumor specificity. This study evaluates its anti-tumor activity, mechanism of action, ability to treat challenging tumors, and safety profile.

Methods: To validate tumor selectivity, V66 or a control antibody were conjugated to a fluorescent tag and injected intravenously into tumor-bearing mice; biodistribution analysis demonstrated selective accumulation in tumors and nuclear localization within tumor cells. V66 was then conjugated to exatecan via a Cleavable Linker. In vitro assays across diverse Cancer cell lines assessed cytotoxicity, DNA damage response (DDR) activation, and TOP1 degradation. In vivo efficacy was evaluated in xenograft models of triple-negative breast Cancer (TNBC) and BRCA1/2-deficient tumors, including intracranial medulloblastoma. These models were used to assess tumor growth inhibition, survival benefit, and blood-brain barrier (BBB) permeability. Toxicity was assessed through a dose-escalation study, with analysis of hematologic parameters, histopathology of major organs, and liver and kidney function tests (ALT, AST, BUN, total protein) following short- and long-term treatment.

Results: V66-exatecan demonstrated potent anti-tumor activity in multiple Cancer cell lines but not on healthy mouse primary fibroblasts, with EC₅₀ values in the low nanomolar range. It induced robust DDR signaling, TOP1 degradation, and bystander killing effects. BRCA1/2-deficient models exhibited enhanced penetration and sensitivity, with up to 17-fold lower EC₅₀ compared to BRCA-proficient controls. In vivo, V66-exatecan significantly inhibited tumor growth and extended survival in both TNBC and BRCA-mutant CNS tumors, including complete regressions and prolonged median survival in BRCA2-deficient models. Toxicology studies revealed no significant hematologic, renal, hepatic, or bone marrow toxicity, even at high or repeated doses.

Conclusions: V66-exatecan represents a next-generation of ADCs that overcomes key limitations of traditional platforms by exploiting exDNA-driven tumor selectivity and ENT2-mediated nuclear delivery. It demonstrates broad therapeutic efficacy and a favorable safety profile, supporting its potential for treating DDR-deficient and hard-to-reach tumors.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12943-025-02462-z.

Antibody-Drug conjugate; BRCA-deficient tumors; DNA damage; Drug delivery system; ENT2; ExDNA; Nuclear penetration; Targeted cancer therapy; Topoisomerase-I; Tumor microenvironment.