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

Figure 2.

(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.

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) 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.