Fig 2: Stimulation of systemic T and NK cell responses by dsIL-18-Fc. Mice bearing established subcutaneous MC38 tumors were injected once intravenously with equimolar doses of WT IL-18 (1 mg/kg) or dsIL-18-Fc (5 mg/kg) and evaluated for T and NK cell activation in blood or tumor-draining lymph nodes after 2 or 4 days (see also online supplemental figures S4 and S5). (a–b) Representative flow cytometry histograms displaying CD44, PD-1, Ki67, and CD62L expression in peripheral blood CD8+ T cells at day 4 (panel a), and associated frequencies of positive cells (panel b). (c) Representative flow cytometry histograms of IFN-γ expression (top panels) and relative IFN-γ mean fluorescence intensities normalized to control group mean (lower panels) in peripheral blood CD8+ T cells, Foxp3− conventional CD4+ T cells, and NK cells. (d) Serum concentrations of IFN-γ, TNF, CXCL9, and CXCL10 after 4 days of treatment. (e–g) Flow cytometry analysis of CD8+ T cells from tumor-draining lymph nodes. Representative plots displaying CD44, PD-1, and Ki67 expression are shown in panel (e), and frequencies of cells positive for Ki67, PD-1, or IFN-γ are quantified in panel (f). Mean frequencies of cells expressing combinations of Ki67 and/or PD-1 are provided in panel (g). Bar charts in panels b, c, d, and f represent mean (±SEM) from five to six mice per group, compared using one-way analysis of variance with Holm-Sidak multiple comparisons test. P values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. Pie charts in panel g represent average frequencies of the indicated phenotypes among CD8+ T cells, and asterisks indicate statistically significant differences (p<0.05) compared with corresponding populations from control-treated mice. All data are representative of two to three independent experiments. dsIL-18, disulfide-stabilized IL-18; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; NK, natural killer; TNF, tumor necrosis factor; WT, wild-type.

Fig 3: Potency, PK, and serum stability of dsIL-18-Fc. (a) In vitro potency of human (left) and mouse (right) WT and engineered dsIL-18 variants was tested in a primary T-cell assay where the IFN-γ production (pg/mL) is plotted against cytokine concentration (pM). (b) The 50% effective concentration (EC50) and the maximum effective concentration (Emax) values of WT and dsIL-18 variants. (c) Pharmacokinetic (PK) profile of mouse WT and dsIL-18-Fc (T44C/L189C). The maximum concentration (Cmax) and the area under the plasma concentration-time curve (AUC) are reported as mean±SD. (d) The stabilites of mouse WT, dsIL-18-Fc (T44C/L189C) and (e) human WT, dsIL-18-Fc (A162C/I185C) are reported as changes in EC50 values over time, following incubation at 37°C in either PBS or serum. Time points include 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 7 days, 14 days, and 21 days. dsIL-18, disulfide-stabilized IL-18; IFN, interferon; IL, interleukin; PBS, phosphate-buffered saline; WT, wild-type.

Fig 4: IL-18 biology, disulfide-stabilized IL-18 (dsIL-18) and its biophysical characterization. (a) Illustration of IL-18 pathway activation. IL-18 first binds to IL-18Rα, and this complex subsequently recruits the IL-18Rβ chain. The dimerization of intracellular TIR domains results in the recruitment of myeloid differentiation primary response 88 (MyD88), interleukin-1 receptor-associated kinase (IRAK1/4) and tumor necrosis factor receptor-associated factor 6 (TRAF6). This results in the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB), leading to the production of pro-inflammatory cytokines such as interferon-γ (IFN-γ). (b) Illustration of the dsIL-18-Fc approach, where the engineered cytokine binds both IL-18Rα and IL-18BP. Created in BioRender. Bulutoglu, (2025) https://BioRender.com/n2qq7ll (c) The yield, aggregation percentage and relative melting temperatures (compared with WT) of dsIL-18-Fc variants. (d) Crystal structures of dsIL-18 (L45C/E192C) (PDB: 9OD7) and dsIL-18 (A162/I185C) (PDB: 9OD9) where the artificially installed disulfide bonds are displayed in magenta. (e) The binding kinetics of WT and engineered IL-18 variants for IL-18Rα and IL-18BP. The equilibrium dissociation constant (KD), the association rate constant (Ka), and the dissociation rate constant (Kd) values are reported as mean±SEM. §KD was calculated from Kd/Ka; KD SEM was propagated from SEM for Ka and Kd values. IL, interleukin; IL-18BP, IL-18 binding protein; WT, wild-type.