Nanobody-Engineered Bispecific IL-18 Mimetics Drive Antitumor Immunity by Engaging CD8+ T Cells 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 cells responses with distinct transcriptomic profiles. Systemic delivery of A4B2-mdFc boosted CD8+ T cells infiltration and activation, demonstrating dose-dependent antitumor efficacy in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Interestingly, PDCD1 and CTLA-4 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 cells activation and IL-18BP resistance, providing the potential application of cytokine-targeting bispecific nanobody monotherapy or in combination with ICIs for cancer immunotherapy.