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. 2023 Jan-Dec;15(1):2236265.
doi: 10.1080/19420862.2023.2236265.

Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition

Britta Lipinski  1   2 , Laura Unmuth  3 , Paul Arras  1 , Stefan Becker  3 , Christina Bauer  1 , Lars Toleikis  3 , Simon Krah  1 , Achim Doerner  1 , Desislava Yanakieva  1 , Ammelie Svea Boje  4 , Katja Klausz  4 , Matthias Peipp  4 , Vanessa Siegmund  3 , Andreas Evers  1 , Harald Kolmar  2 , Lukas Pekar  1 , Stefan Zielonka  1   2
Affiliations

Generation and engineering of potent single domain antibody-based bispecific IL-18 mimetics resistant to IL-18BP decoy receptor inhibition

Britta Lipinski et al. MAbs. 2023 Jan-Dec.

Abstract

Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rβ. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.

Keywords: Antibody engineering; IL-18; IL-18 binding protein; IL-18BP; VHH; bispecific antibody; cytokine mimetic; single domain antibody; surrogate agonist; valencies; yeast surface display.

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Conflict of interest statement

BL, PA, LU, LP and SZ filed a patent application based on this work. In addition, BL, PA, LU, LP, SK, SB, LT, VS, AE, and SZ are employees at Merck Healthcare KGaA. Besides, this work was conducted in the absence of any further commercial interest.

Figures

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.
Figure 1.
Overall strategy for the generation of tailor-made cytokine mimetics based on sdAb-derived bispecifics and YSD-enabled antibody discovery.
(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.
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) 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.
Figure 3.
Bispecific (1 + 1) surrogate agonists trigger IFN-γ release on PBMCs isolated from healthy donors.
Figure 4.
Figure 4.
Antibody Engineering enables the generation of IL-18 mimetics with augmented agonism capacities.
See this image and copyright information in PMC

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