CD44-Targeted Antibody-Drug Conjugates Suppress Ovarian Tumor Progression and Remodels Cancer Stem Cell-Associated...

Cluster of differentiation 44 (CD44) is highly expressed in ovarian cancer (OC) and plays a critical role in cancer stem cell (CSC) maintenance, tumor initiation, and chemoresistance, making it an attractive therapeutic target. Herein, a CD44-targeted antibody-drug conjugate (ADC), CD44-VC-DOX, is developed by conjugating doxorubicin (DOX) to an anti-CD44 monoclonal antibody using a cathepsin-cleavable MC-VC-PAB linker for selective intracellular drug release in CD44-positive tumor cells. Physicochemical characterization confirms successful conjugation, high stability, and an optimal drug-to-antibody ratio. CD44-VC-DOX exhibit selective binding and rapid internalization in CD44-overexpressing OC cells resulting in potent cytotoxicity, apoptosis induction, and G2/M cell-cycle arrest. Importantly, CD44-VC-DOX effectively suppresses CSC-associated phenotypes, including tumorsphere formation, tumor-initiating frequency, CD44⁺CD133⁺ CSC populations, ALDH-positive cells, and stemness-related gene expression. In addition, CD44-VC-DOX reduces hypoxia-associated HIF-1α expression, indicating disruption of the CSC-supportive tumor microenvironment. In vivo, CD44-VC-DOX demonstrates efficient tumor targeting, significant tumor growth inhibition, and minimal systemic toxicity. Transcriptomic profiling further reveals coordinated suppression of CSC-associated and oncogenic pathways, including PI3K/AKT, MYC signaling, E2F targets, DNA repair, and stemness-related transcriptional programs, supporting effective depletion of tumor-initiating CSC populations and reduced tumor adaptability. Collectively, these findings highlight CD44-VC-DOX as a promising precision therapeutic strategy for targeting CSC-driven OC.