https://orcid.org/0000-0001-9259-0965
Figures & data
Figure 1. Overall strategy for the generation of tailor-made cytokine mimetics based on sdAb-derived bispecifics and YSD-enabled antibody discovery.
IL-18 binds to the IL-18R that is composed of two subunits, IL-18Rα and IL-18Rβ. However, IL-18 is efficiently neutralized by IL-18BP. In contrast to this, bispecific IL-18 mimetics based on sdAbs bind to the IL-18 receptor without being inhibited by IL-18BP. (B) FACS plots showing the enrichment of VHHs against both IL-18R subunits from yeast surface display libraries based on IL-18R immunized Llamas and Huarizos. (C) Multiple sequence alignment showing a broad diversity of isolated clones against both receptor subunits. The highest sequence variation can be found in the different CDRs, mainly in CDR3.
(A) Left: IL-18 (blue) triggers IL-18 R downstream signaling by consecutive binding to IL-18 Rα (yellow) followed by IL-18 Rβ (lime) recruitment. IL-18BP (dark gray) inhibits IL-18 by high-affinity binding and hence, by blocking the IL-18/IL-18 Rα interaction. Right: Camelid-derived sdAbs enable targeting IL18-Rα (orange) and IL-18 Rβ (green). Upon reformatting into an IgG-like bispecific, resulting cytokine mimetics cross-link the IL18R subunits and elicit down-stream signaling. Essentially, generated IL-18 mimetics are resistant to inhibition by IL-18BP. Structural visualization was generated with PyMOL software version 2.3.0, based on PDB entries 3WO4 and 7AL7, structural modeling as described in the methods section and modified using www.biorender.com. (B) Immunization of camelids followed by YSD facilitate the enrichment of sdAbs specific to (rh) IL-18 Rα and (rh) IL-18 Rβ. One sublibrary was generated for each specimen (huarizo and llama) and sorted separately. In the first round of selection, a mixture of both (rh) receptor subunits was exploited at a concentration of 250 nM. In the subsequent second sorting round, enriched libraries were sorted separately against each antigen at 250 nM. A two-dimensional sorting strategy was applied to select for full-length VHH display in addition to antigen binding. Percentage of cells in sorting gates are shown. Plots show 5 × 104 events. (C) Graphical alignments of 55 unique sdAb clones addressing IL-18 Rα (top) and 101 independent clones targeting IL-18 Rβ (bottom) retrieved from YSD library sorting. Complementarity-determining regions (CDRs) are highlighted. Red bars indicate high sequence diversity at the amino acid level and green bars represent high sequence conservation at a given position. Alignment conducted with MUSCLE alignment tool using Geneious Prime 2021.1.1.
Figure 2. Combinatorial reformatting of monospecific (1 + 0) SEEDbodies into strictly monovalent (1 + 1) bsAbs enables the identification of IL-18 mimetics with attenuated capacities to trigger NFκB reporter activity on IL-18 reporter cells.
(A) Concentration-dependent activation of HEK-Blue™ reporter cells by bispecific sdAb-based cytokine mimetics. (B) Heatmap of NFκB reporter activitation elicited by bispecifc IL-18 mimetics showing multiple receptor agonist colored in green, minimally active mimetics in yellow, inactive entities in red as well as molecules with inadequate expression yields or purities in grey.
(A) HEK-Blue™ reporter cells were incubated with increasing concentrations of reformatted bsAbs, as exemplarily shown for IL18R_VHHα2β15, IL18R_VHHα8β15, IL18R_VHHα2β17, IL18R_VHHα8β17 and IL18R_VHHα1β16. Secreted embryonic alkaline phosphatase activity was monitored by determining the OD640. Reporter activity was normalized to maximal IL-18 read-out. As negative control, (rh) TNF was used. Graph shows one respective screening experiment. (B) Heatmap of NFκB reporter activitation elicited by combinatorial reformatted (1 + 1) bsAbs. Molecules failing initial quality control (target monomer peak in SEC < 86% post-protein A purification given in dark gray, functionally inactive bsAbs shown in red, minimally active surrogate agonists (NFκB reporter activitation < 15% compared to (rh) IL-18 at 1 nM or EC50 ≥0.1 nM) in yellow and moderately active IL-18 mimetics (NFκB reporter activitation ≥ 15% compared to (rh) IL-18 at 1 nM or EC50 <0.1 nM) depicted in green.
Figure 3. Bispecific (1 + 1) surrogate agonists trigger IFN-γ release on PBMCs isolated from healthy donors.
(A) Bispecific IL-18 surrogate agonists trigger IFN-γ production on PBMCs of multiple donors at a fixed concentration of 100 nM. (B) Selected bispecific IL-18 mimetics elicit a dose-dependent IFN-γ on PBMCs of multiple healthy donors with varying potencies.
(A) IFN-γ production of PBMCs stimulated with bsAbs at a fixed concentration of 100 nM or with (rh) IL-18 at 1 nM. Experiments were performed 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 10 different donors. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.IL18R_VHHα1β16 was used as negative control (given in red). Four leading candidates used for further characterization shown in green, purple, blue, and orange. (B) TOP4 candidates evoke a dose-dependent IFN-γ read-out on PBMCs 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 10 independent experiments p[,//’/l;\]=\][l; are shown.
Table 1. Binding kinetics and functional properties of the herein generated four leading bispecific IL-18 mimetics.
Figure 4. 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.
(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.
Table 2. Biophysical, biochemical, and functional attributes of engineered cytokine mimetic formats.
Supplemental material