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. 2023 Jul 19;15(1):2236265. doi: 10.1080/19420862.2023.2236265

Figure 4.

(A) Different format architectures of one selected bispecific IL-18 mimetic including multivalent tandem-arrangements as well as VHHs replacing VH and VL domains in an IgG. (B) Arranging IL-18R binding VHHs in tandem enables a more efficient IFN-γ response on human PBMCs isolated from healthy donors at two fixed concentrations (10 nM and 1 nM). (C) Tandem arrangements of one selected bispecific IL-18 mimetic are significantly more potent in triggering a functional IFN-γ release on PBMCs compared with a strictly monovalent surrogate agonist in a dose-response curve. (D) IFN-γ release on PBMCs triggered by (rh) IL-18 is efficiently inhibited in a concentration-dependent manner by (rh) IL-18BP, whereas IFN-γ response elicited by engineered bispecific IL-18 mimetics is not negatively affected by (rh) IL-18B.

Antibody Engineering enables the generation of IL-18 mimetics with augmented agonism capacities.

(A) Schematic depiction of main different bispecific antibody architectures that were constructed within this work. Fusion of an anti-IL18Rα VHHα2 (orange) to the hinge region of the AG chain of the SEEDbody as well as engraftment an anti-IL18Rβ VHHβ15 (green) onto the GA chain results in the initially generated (1 + 1) format IL18R_VHHα2β15. Replacing the VH and VLλ of an effector silenced IgG by VHHα2 and VHHβ15, respectively, facilitates the generation of the IL18R_sdIgGα2β15 architecture (2 + 2). Within the IL18R_tanVHHα2β15 design (2 + 2), VHHα2 and VHHβ15 are arranged in tandem (from N-terminus to C-terminus) and separated by a five amino acid Gly4Ser linker. The tandem is fused to the hinge region of an effector silenced IgG1 Fc fragment. Of note, also the opposite orientation was constructed (VHHβ15 followed by VHHα2, IL18R_tanVHHβ15α2). In addition, all four molecules were also produced harboring the E430G mutation for on-target hexamerization. (B) Distinct surrogate agonist formats of the same paratopes (VHHα2 and VHHβ15) display differential properties in eliciting a functional IFN-γ response on human PBMCs isolated from healthy donors at fixed concentrations. Experiments were performed at two different concentrations (10 nM and 1 nM) in the presence of 10 ng/mL (rh) IL-12. Graph shows box and whisker plots as superimpositions with dot plots of IFN-γ release of six different donors. ****p < 0.0001, **p < 0.01, *p < 0.05. (C) Surrogate agonists arranged in tandem (IL18R_tanVHHα2β15 and IL18R_tanVHHβ15α2) elicit enhanced IFN-γ production in terms of potencies and magnitude on PBMCs isolated from healthy donors, resulting in a variant with increased potencies compared with (rh) IL-18. All experiments were performed in the presence of low dose (rh) IL-12 (10 ng/mL). IL18R_VHHα1β16 as negative control shown in red was used at a fixed concentration of 1 µM. Mean values ± SEM of 13 independent experiments are shown.****p < 0.0001(D) Potency augmented tandem IL-18 mimetics are resistant to inhibition by (rh) IL-18BP, whereas (rh) IL-18 is efficiently blocked from signaling. PBMCs of healthy human donors were stimulated either with (rh) IL-18 or IL18R_tanVHHα2β15 and IL18R_tanVHHβ15α2 at a fixed concentration of 0.5 nM in the presence of (rh) IL-12 (10 ng/mL) and different concentrations of (rh) IL-18BP. Five independent experiments were performed and mean values ± SEM are shown. ****p < 0.0001,***p < 0.001.