CD44‐Targeted Antibody‐Drug Conjugates Suppress Ovarian Tumor Progression and Remodels Cancer Stem Cell‐Associated Molecular Programs

ABSTRACT 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.