Dual-Targeting Multivalent Aptamer-Drug Hybrids for Synergistic Cancer Immunotherapy.

Targeted drug conjugates (TDCs) have transformed cancer therapy by enabling selective delivery of cytotoxic agents, yet most existing designs rely on single-antigen targeting and single-payload architectures that limit efficacy in heterogeneous tumors and restrict opportunities for integrating orthogonal mechanisms of action. Here, we report circular, dual-targeting multivalent aptamer-drug hybrids (Dualo-mvApDHsD/S) that codeliver doxorubicin (Dox) and STING agonist (diABZI) for synergistic chemo-immunotherapy. Built on a programmable DNA scaffold with defined valence and high loading capacity, the Dualo-mvApDHsD/S simultaneously engage c-Met and CD71 to enhance tumor-specific uptake through heteromultireceptor-mediated endocytosis, achieving efficient intracellular delivery and robust tumor accumulation in vivo. Within tumor cells, Dox induces genotoxic stress and potent immunogenic cell death, while diABZI activates cGAS-STING signaling to amplify type I interferon responses. This coordinated action remodels the immunosuppressive tumor microenvironment, promoting dendritic cell recruitment and activation, expanding IFN-responsive macrophages and conventional dendritic cells, and driving the proliferation and functional maintenance of cytotoxic CD8+ T cells. Single-cell RNA and TCR sequencing revealed increased TCR diversity, reduced terminal exhaustion, and strengthened effector differentiation in response to combination therapy. Notably, Dualo-mvApDHsD/S synergize with PD-1 blockade to achieve durable tumor eradication and long-term protection. These findings establish multivalent aptamer-drug hybrids as a versatile platform for multitarget, multipayload precision therapeutics and highlight their potential for next-generation TDC design.