Targeting intrinsically disordered nuclear protein 1 (NUPR1) with single-domain antibodies alleviates triple-negative breast cancer (TNBC) progression in vivo.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype that currently lacks effective targeted therapies. Transcriptional co-regulator nuclear protein 1 (NUPR1) has been identified as a key stress-adaptive disordered protein that promotes tumor progression and therapy-induced resistance. In this study, we developed a robust high-throughput platform integrating in situ proximity ligation assay followed by DNA sequencing (isPLA-seq), NanoBiT assays, and C-degron degradation validation to screen for functional single-domain antibodies (sdAbs) targeting NUPR1. Consequently, sdAb#07.81 emerged as a lead candidate, demonstrating strong binding affinity and the ability to degrade endogenous NUPR1 in TNBC cells. Functional assays confirmed that sdAb#07.81 suppressed TNBC cell growth, induced premature senescence, and derepressed ferroptosis. In vivo validation using a 4T1 cell-derived fully immunocompetent murine model further established its therapeutic efficacy, with significant reductions in tumor size, NUPR1 expression, and cell proliferation. These findings highlight sdAb#07.81 as a promising therapeutic agent and validate the platform's effectiveness for addressing intracellular disordered targets like NUPR1. This work underscores the potential of sdAbs as a cancer therapeutic and provides a foundation for advancing sdAb#07.81 into preclinical and clinical development to address the critical unmet needs of TNBC treatment.