De novo design of miniprotein antagonists of cytokine storm inducers

Abstract

Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.

Fig. 1. IL-6 and IL-1 in cytokine and antagonist design strategy.

a Signaling pathway of IL-6 and IL-1 in cytokine storm. b IL-6R blockade design strategy by directly competing IL-6 binding to IL-6R at site-I. c GP130 blockade design strategy by interfering with the IL-6/IL-6R complex binding to GP130 at site-II. d IL-1R blockade design strategy by directly competing IL-1b binding to IL-1R at the intersection of domain1 (D1) and doamin2 (D2).

Fig. 2. Designed minibinder antagonists and characterization.

a, e, and i Design model of minibinder in complex with targets. a IL-6R (PDB: 1P9M, 1N26); e GP130 (PDB: 1P9M); i IL-1R (PDB: 1IRA). The major binding sites/hotspots were colored yellow. b, f, and j The zoomed interface of the initial IL-6R minibinder design model colored by conservation calculated based on site mutagenesis screening. The designed minibinders are colored based on entropy, where blue indicates the conserved position and red indicates the less conserved position. c, g, and k Binding affinity measurement using BLI. 50 nM biotinylated targets were captured on SA-tips and incubated with given concentrations of minibinders. Binding affinities were estimated using the Octet ForteBio software. d, h, and l CD spectra before and after melting. Source data are provided as a Source Data file (raw data for c, g, and k are provided in the raw data file zenodo.org (accession code: 12797779) due to file size).

Fig. 3. Cytokine signaling inhibition effects of designed antagonists.

a, b Measurement of  antagonism of STAT3 signaling by the IL-6R and GP130 antagonists using phosphoflow. THP-1 cells (a) or HUVEC cells (b), which do not express IL-6R, were titrated with increasing concentrations of IL-6 alone or with 100 nM IL-6Rmb10 or GP130mb33 (a) or preincubated with 200 nM soluble IL-6R for trans-signaling followed by addition of IL-6 alone or with 500 nM IL-6Rmb10 or GP130mb33 (b). c Antagonism of IL-6 induced proliferation. Starved TF-1 cells were incubated alone or with 1 uM of IL-6Rmb10 or GP130mb33 and stimulated with three different concentrations of IL-6 for 2 days before assessing cell viability. The P value is calculated by an unpaired two-tailed t-test. d Measurement of antagonism of P38 signaling by IL-1R antagonism measured by phosphoflow. THP-1 cells were titrated with increased concentrations of IL-1Rmb81 in the presence of 10 nM IL-1b. For a–d, the mean values were calculated based on biological replicates with sample size n = 3 and error bars represent SEM. The IC50s were measured by non-lin fit using GraphPad Prism 8.0.2. Source data is provided as a Source Data file.

Fig. 4. Crystal structures of designed antagonists.

a Left, alignment of design model of GP130mb_original (before optimization) in complex with GP130 on the experimentally determined crystal structure of the GP130/GP130mb33 complex. The aligned complex rmsd using pymol2 is indicated. Right, zoom in on the interface of the aligned complex model. b, c Superimpositions of the experimentally solved monomer structures of designed antagonists on the design models (before optimization); the targets are shown as gray surfaces. The aligned monomer rmsds using pymol2 are indicated. b IL-6Rmb6 structure aligned to IL-6Rmb_original in complex with IL-6R (design model). c IL-1Rmb80 structure aligned to IL-1Rmb_original in complex with IL-1R (design model).

Fig. 5. Functional study of designed antagonists in the human organoid model.

a Treatment regimen for injured hCOs. b FAC quantification. Error bars are from n = 16  biological replicates. c Calculated ratio of Vimentin vs α-SA in the immunofluorescent staining assay. The error bar is shown with biological replicate n = 5. d Immunofluorescent staining of hCOs or IL-1β stimulated hCOs on Day 4 (green = α-SA, red = Vimentin, and blue = DAPI). The scale bar represents 50 μm for each image. e Cytokine multiplex comparison among IL-1R inhibitors, IL-6Rmb6, and GP130mb33 inhibitors. Results were represented as fold change of cytokines in the Day 4 supernatant as compared to hCOs. The error bar is shown with biological replicate n = 4. For b, c, and e data were presented as mean values +/− SEM, and P values were calculated via an ordinary one-way ANOVA test. For P values, ns represents P > 0.05, *represents P < 0.05, **represents P < 0.01, ***represents P < 0.001 and ****represents P < 0.0001. Source data are provided as a Source Data file.