BivalentOX40 Aptamer and CpG as Dual Agonists forCancer Immunotherapy

Cancer immunotherapy has revolutionized cancer treatment by harnessing the body’s immune system to recognize and attack tumors. Over the past 25 years, the use of blocking antibodies has fundamentally transformed the landscape of cancer therapy. However, despite extensive research, agonist antibodies targeting costimulatory receptors such as ICOS, GITR, OX40, CD27, and 4-1BB have consistently underperformed in clinical trials over the past 15 years, failing to meet the anticipated success. One reason the agonist antibodies failed is that researchers escalated the dose to the highest tolerable level, which can lead to cell exhaustion, especially when used as a single agent. In this study, we introduced novel <i>in situ</i> therapeutic agents by combining a bivalent RNA aptamer of OX40, biR^OX40, which binds to two copies of the OX40 receptor as an agonist, with CpG, a toll-like receptor 9 (TLR9) immune stimulator. These agents were specifically designed for lymphoma treatment, with the dose reduced to the lowest bioactive amount to maximize efficacy while minimizing potential side effects. BiR^OX40 and CpG exhibited a dual immune activation effect and demonstrated a synergistic response even at extremely low dose of 0.32 mg/kg (5.75 μg per mouse) for biR^OX40 and moderate dose of 1.39 mg/kg (25 μg per mouse) for CpG, resulting in remarkable antitumor efficacy. This effect was achieved through the promotion of intratumoral CD8+ T cell proliferation and cytokine secretion, inhibition of regulatory T cell (Treg) proliferation, and enhanced generation and proliferation of memory T cells in immune organs. The agonistic effects of these reagents led to tumor regression not only at the treated sites but also at distant, nontreated locations in the animal models. This outcome highlighted the induction of a robust systemic antitumor immune response, which effectively suppressed tumor recurrence. This <i>in situ</i> combination therapy, utilizing low-dose biR^OX40 alongside CpG, offers a straightforward and widely applicable strategy to enhance immune responses in cancer immunotherapy. This approach overcomes the limitations of high-dose single-agent anti-OX40 therapies (whether antibodies or aptamers), including immune cell exhaustion and diminished efficacy.