Nanobody-engineered bispecific IL-18 mimetics drive antitumor immunity by engaging CD8(+) T cell and evading IL-18BP in preclinical models.

Cytokines are promising in cancer immunotherapy, but their pleiotropic effects limit specificity and clinical utility. Through binding to IL-18Rα and IL-18Rβ, interleukin-18 (IL-18) stimulates innate lymphocytes and effector T cells for antitumor immunity. However, clinical trials of recombinant IL-18 have been hampered by IL-18 binding protein (IL-18BP), a secreted high-affinity decoy receptor. Here, we developed decoy-resistant bispecific nanobodies that maintain IL-18 signaling potential. Based on agonistic nanobodies targeting IL-18Rα and IL-18Rβ, bispecific nanobody A4B2-mdFc effectively enhanced CD8(+) T cell responses with distinct transcriptomic profiles. Systemic delivery of A4B2-mdFc boosted CD8(+) T cell infiltration and activation, demonstrating dose-dependent antitumor efficacy in cell-line-derived xenograft and patient-derived xenograft models. Interestingly, PDCD1 and CTLA4 expressions were drastically increased on CD8(+) T cells when treated with A4B2-mdFc. Injection of A4B2-mdFc significantly improved the antitumor efficacy of immune checkpoint inhibitors (ICIs) targeting both PD-1 and CTLA-4. Our findings demonstrated that nanobody-based bispecific IL-18 mimetics elicited superior antitumor activity via CD8(+) T cell activation and IL-18BP resistance, providing the potential application of cytokine-targeting bispecific nanobody monotherapy or in combination with ICIs for cancer immunotherapy.