AI-designed PD-1/IL-18v bispecific antibody overcomes PD-1 resistance and drives potent antitumor responses in refractory models

Cytokines are powerful regulators of antitumor immunity, yet their therapeutic use is limited by short in vivo half-life and systemic inflammatory toxicity. Interleukin-18 (IL-18) is particularly attractive because it strongly activates NK cells and CD8⁺ T cells, the main tumor-killing lymphocytes. Recent work has shown that antibody-cytokine conjugates such as IL-2 and IL-15 fused to anti-PD-1 antibodies can localize cytokine activity to PD-1-rich tumors and reinvigorate impaired anti-tumor immunity while avoiding systemic exposure. Motivated by this strategy, we applied our AI-driven protein therapeutics design platform, GaluxDesign, to create a next-generation IL-18 variant (IL-18v) optimized for tumor-restricted activity.Using GaluxDesign, we engineered an IL-18v that abolishes IL-18BP binding while preserving attenuated biological activity, even though its affinity for IL-18 receptor alpha (IL-18Ralpha) was intentionally minimized. IL-18v showed a >10°C increase in thermal stability over wild-type IL-18 and fully escaped IL-18BP-mediated inhibition. To confine IL-18v activity to the tumor microenvironment (TME) and minimize systemic toxicity, we generated a PD-1/IL-18v bispecific antibody for cis-targeted delivery of IL-18v to PD-1⁺ T cells. Although free IL-18v has minimal IL-18Ralpha binding, its potency was restored when conjugated to an anti-PD-1 antibody, enabling PD-1-dependent cytokine activation. The bispecific molecule exhibited minimal activity in PD-1⁻ IL-18 reporter cells and NK cells, but showed markedly enhanced signaling activity in PD-1⁺ IL-18R reporter cells and, notably, an >800-fold increase in immune activity specifically in PD-1⁺ NK92 cells, supporting a mechanism in which IL-18v remains inert peripherally but becomes activated only in PD-1-rich TMEs.In human PD-1 knock-in mice, the PD-1/IL-18v bispecific antibody incorporating a mouse IL-18v surrogate induced >90% tumor regression in the PD-1-responsive MC38 syngeneic model, whereas anti-PD-1 monotherapy showed minimal effect. PD-1/IL-18v also remodeled the TME into a highly inflammatory, antitumor state, as confirmed by immune profiling. In PD-1-less responsive models such as CT26 and in strongly PD-1-refractory tumors like B16-F10, the bispecific surrogate achieved >90% tumor growth inhibition despite complete resistance to PD-1 blockade. Notably, body weight remained stable over repeated treatments, indicating minimal systemic cytokine toxicity.Collectively, these results demonstrate that the AI-engineered PD-1/IL-18v bispecific antibody elicits provides potent PD-1-dependent IL-18 signaling with tumor specificity while limiting systemic adverse effects. The combination of IL-18BP resistance, diminished IL-18Ralpha affinity and PD-1-restricted activation creates a compelling basis for next- generation cytokine-based cancer immunotherapy.