Programmable multivalent aptamer-drug conjugates enable stable tumor targeting and enhanced therapy for advanced...

The therapeutic potential of aptamer-drug conjugates (APDCs) is constrained by challenges in achieving high in vivo stability, strong target affinity, and binding robustness under blood flow-induced mechanical stress. To address these limitations, multivalent APDCs have been actively explored; however, most reported designs rely on heterogeneous assemblies, and their binding stability under mechanical perturbations remains poorly characterized. Here, we report a programmable and structurally defined multivalent APDC, termed Multi-MMAE, constructed via hybridization chain reaction (HCR)-mediated DNA assembly. This architecture enables tunable modulation of aptamer multivalency and drug incorporation, allowing multivalent presentation of both the PTK7-targeting aptamer Sgc-8 and the cytotoxic payload monomethyl auristatin E via a cleavable VC-PAB linker. Compared with monovalent Sgc-8-MMAE, Multi-MMAE exhibits enhanced serum stability, ∼11.5-fold higher binding affinity, improved binding stability under flow-mimicking perturbations, and increased cellular uptake and intracellular drug delivery. At equivalent MMAE doses, Multi-MMAE demonstrates superior antitumor efficacy and survival benefits in both orthotopic and experimental lung colonization bladder cancer mouse models, accompanied by prolonged circulation, enhanced tumor retention relative to Sgc-8-MMAE, and reduced systemic toxicity compared with free MMAE. Mechanistic studies further reveal that structural multivalency modulates intracellular trafficking pathways and pharmacodynamic behavior. Collectively, this work establishes a modular aptamer-drug conjugate strategy for advancing APDC-mediated cancer therapy.