Aptamer-enhanced immunotherapy targeting Non-Small Cell Lung Cancer cells: A conceptual approach towards T-cell mediated therapy

Despite clinical advances with immune checkpoint inhibitors targeting the PD-1/PD-L1 axis, therapeutic response remains limited, as nearly 60% of PD-L1–positive patients fail to benefit from anti-PD-L1 monoclonal antibody therapy. Aptamers are short, single-stranded DNA or RNA oligonucleotides that selectively bind to target molecules with high affinity and specificity. Compared to monoclonal antibodies, aptamers offer several advantages, including lower production cost, faster synthesis, improved thermal stability, longer shelf life, and minimal immunogenicity, making them attractive candidates for targeted cancer therapy. In this study, we designed DNA aptamers specific to the immune checkpoint receptor PD-L1, which is overexpressed on non-small cell lung cancer (NSCLC) cells and serves as a key regulator of tumour-induced immune suppression. Using comprehensive in silico screening, structural modelling, and interaction analysis, aptamer63 was identified as the strongest PD-L1 binder based on predicted thermodynamic stability and molecular interaction energy profiles. To experimentally validate its functional potential, aptamer63 was fluorescently labelled with TAMRA and visualised under a fluorescence microscope, confirming its binding to the cell surface of PD-L1–expressing NCI-H460 NSCLC cells. Subsequent in vitro assays assessed the immunomodulatory effect of aptamer63 in the presence of activated T lymphocytes. MTT and trypan blue dye exclusion assays demonstrated a dose-dependent reduction in tumour cell viability, with significant inhibition observed at 100nM and 200nM. Hoechst 33342/propidium iodide dual staining confirmed increased apoptotic cell populations, showing the highest apoptosis rate at 100nM. Flow cytometry further revealed a marked decrease in viable cell count across all tested concentrations, with the greatest reduction again observed at 100nM. Collectively, these results indicate that aptamer63 enhances T cell–mediated cytotoxicity against NSCLC cells, with maximum antitumour efficacy at 100nM. Overall, these findings establish aptamer63 as a promising PD-L1-blocking immunotherapeutic candidate for NSCLC and demonstrate a rational design pipeline for developing next-generation aptamer-based immune checkpoint inhibitors.