Anti-CD24 antibody-nitric oxide donor conjugates bearing a self-bioorthogonal cleavable linker

Available online 30 July 2025

Author links open overlay panel, , , , , , , , , , , Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy predominantly managed via chemotherapy. Our clinical sample analysis revealed a significant correlation between elevated CD24 expression in TNBC tumor cells and patient survival rates. We developed a novel antibody–drug conjugate (ADC), named HN03, consisting of an antibody with engineered cysteines for site-specific conjugation with a low toxic nitric oxide (NO) precursor as its payload through a novel Pt(IV)-mediated bioorthogonal self-cleavable linker. HN03 specifically targets tumor cells expressing high levels of CD24, concurrently generating cisplatin and releasing NO upon activation. HN03 also exhibited potent in vitro and in vivo antitumor activity. It significantly reduced tumor growth at various doses, prevented tumor metastasis, with markedly lower toxicity than traditional chemotherapy agents. We found that a key mechanism of its action involved inducing apoptosis and endoplasmic reticulum stress, substantially decreasing the number of M2-type macrophages. Overall, HN03 stands out as a promising therapeutic option for TNBC, offering a targeted treatment with reduced side effects and the potential for improved outcomes. Furthermore, using Pt(IV) in the linker and an NO precursor as the payload enhances the versatility of the Antibody-NO donor Conjugate (ANC), offering new avenues for the design of the next generation of ADCs.

Graphical abstractThis study designed a site-specific conjugate HN03, consisting of a cysteine-engineered CD24 antibody and a bioorthogonal catalysis-based NO donor mediated by Pt(IV). By the incorporating Pt(IV) into the linker, which can bio-orthogonally activate the payload NO precursor, the versatility of the ANC is expanded, opening up new opportunities for the development of future ADCs.