Acidic pH-responsive and dissociative dimeric CpG oligodeoxynucleotides for enhanced cancer immunotherapy

In cancer immunotherapy, the immune system is stimulated to specifically recognize and eliminate malignant cells while minimizing harm to normal cells. One widely investigated potential immunotherapeutic agent is CpG oligonucleotides (CpG ODNs), short single-stranded DNA molecules with the ability to stimulate both innate and adaptive immune responses by binding to Toll-like receptor 9 (TLR9) within endosomal membranes. However, their short half-life in systemic circulation presents a significant challenge to achieving therapeutic efficacy. Herein, we designed and developed an acidic pH-responsive, dissociative dimeric CpG ODN nanostructure, termed CpG i-dimers, to improve serum stability and enhance immune responses for effective cancer immunotherapy. The CpG i-dimers comprise a central DNA duplex containing i-motif DNA-forming sequences, with CpG ODNs attached as overhangs at each 5' end of the duplex. Upon internalization into acidic endosomes, the low pH induces a structural transition of the i-motif sequences within the CpG i-dimer, leading to its dissociation into two active CpG ODN molecules. Notably, this pH-responsive dissociation significantly increases the local concentration of CpG ODNs available for TLR9 binding, thereby amplifying downstream signaling. Compared to CpG dimers lacking i-motif DNA-forming sequences, CpG i-dimers markedly suppressed tumor cell proliferation and growth in tumor-bearing mice by eliciting robust immune responses, including enhanced pro-inflammatory cytokine secretion, M1 macrophage polarization, antibody production, and T cell differentiation. The developed CpG i-dimers represent a simple yet highly efficient platform that bridges smart DNA nanotechnology with practical cancer immunotherapy.