Membrane-Anchored Dual-Targeting Aptamer Engagers for Programmable T Cell Redirection in Solid and Hematological Cancers

Abstract Inefficient immune synapse formation limits the therapeutic efficacy of T cell-based immunotherapy in both solid tumors and hematological malignancies. Here, we report a fully synthetic, membrane-anchored dual-targeting aptamer engager, AptE 2, that acts as a programmable artificial immunological interface to redirect cytotoxic T cells toward cancer cells without genetic engineering. Constructed entirely from DNA aptamers through optimized bioorthogonal conjugation and enzyme-mediated ligation, AptE 2 integrates multivalent tumor recognition, T cell binding, enhanced nuclease resistance, and prolonged membrane anchoring within a single modular scaffold. In colorectal cancer models, AptE 2 promoted stable T cell–tumor cell engagement, enhanced cytotoxicity and cytokine secretion, and drove pronounced tumor regression with increased intratumoral CD8+ T cell infiltration and minimal systemic toxicity. Importantly, AptE 2 also demonstrated broad applicability in leukemia. In CCRF-CEM cells, AptE 2 exhibited strong surface binding, sustained membrane retention for up to 6 h, and superior resistance to wash-off compared with monovalent aptamers and bivalent controls. AptE 2-prearmed TALL-104 cells markedly enhanced leukemia cell clustering, increased cell lysis to over 90% under optimized coculture conditions, and amplified IFN-γ secretion. In a disseminated CCRF-CEM-Luc leukemia mouse model, AptE 2-mediated T cell therapy suppressed systemic leukemia progression, improved peripheral hematological profiles, and showed favorable tolerability without detectable major organ injury. These findings establish AptE 2 as a generalizable, nonprotein-based and nonviral immune engager platform for programmable T cell redirection across both solid and hematological cancers.