Preclinical proof of principle for orally delivered Th17 antagonist miniproteins

Abstract

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

Keywords: IL-17; IL-23R; Th17; autoinflammation; computational protein design; inflammatory bowel disease; oral biologics; protein engineering.

Figure 1.. Computational design of IL-23R and IL-17A minibinders.

(A) Minibinder 23R-1 was designed to bind IL-23R domain D1 at the IL-23p19 interaction surface, and all designs incorporate native hotspot W156. (B) Minibinder 17–1 was designed to bind IL-17A at site 1 of the IL-17RA interaction surface and incorporates de novo hotspots (purple) that mimic native hotspots from IL-17RA (dark gray). (C) The relative affinity of each mutation was determined using deep mutational scanning. The enrichment (blue) or depletion (red) of each mutation, depicted in 2D heatmaps, represents its impact on affinity relative to the original minibinder sequence (set to 0, white). Positional conservation scores are depicted in a 1D heatmap from minimum (light gray) to maximum (dark gray) per design. Asterisks indicate native and de novo hotspots. See Figure S1 for binding data and Figures S3 and S6 for deep mutational scanning heatmaps of additional IL-23R and IL-17A computationally designed minibinders and peptides.

Figure 2.. In vitro potency and stability of the IL-23R minibinders.

(A) Minibinders block IL-23-mediated cell signaling in an engineered IL-23 reporter cell line. Representative curves are shown above (n≥2), and IC50 values reported as mean ± SD of at least two independent experiments (N≥2). (B) The binding affinities of 23R-72 and 23R-91 were determined using SPR. (C) 23R-1 and derivatives, and (D) 23R-2 and derivatives were denatured with heat and/or chemical denaturant guanidinium hydrochloride (Gdn), and helicity (signal at 222 nm) was monitored using circular dichroism. Signal is plotted as a fraction of reference sample (0 M Gdn at 25ºC). (E) Molecular design and optimization workflow. See Figures S1 and S3 for binding data and Figure S4 for biophysical characterization of IL-23R minibinder variants.

Figure 3.. In vitro potency and stability of the IL-17 minibinders.

(A) Minibinders block cell signaling mediated by hIL-17A (left), hIL-17F (middle), or hIL-17A/F (right) in an engineered IL-17 reporter cell line. Representative curves are shown above (n≥2), and IC50 values are reported as mean ± SD of at least two independent experiments (N≥2; ND = no data). (B) The binding affinity of lead IL-17A minibinder 17–53 was determined using BLI. We note that the instrument (ForteBio Octet RED96) is not sensitive enough to accurately determine picomolar KDs, but the data nonetheless indicate the very high affinity and slow dissociation rate of 17–53. Data plotted are representative of three independent experiments (N=3). (C) Each minibinder was denatured with heat or chemical denaturant guanidinium hydrochloride (Gdn), and helicity (signal at 222 nm) was monitored using circular dichroism. Signal is plotted as a fraction of reference sample (0 M Gdn at 25°C). (D) Molecular design and optimization workflow. See Figure S1 and S3 for binding analysis and Figure S2 for potency and stability data for other IL-17A minibinder variants.

Figure 4.. In vitro and in vivo GI stability of IL-23R minibinders.

(A) Each minibinder or control protein V565, an oral nanobody in development for IBD, were digested in SIF or SGF at 37°C for up to 24 hours (N≥2). In some instances, indicated by white spaces, lanes were isolated in order to maintain consistent order of ladder and samples. (B) SIF and SGF digests were sampled at the indicated timepoints, diluted to 10 nM minibinder (assuming no degradation), and BLI was used to measure residual binding to hIL-23R (N≥2). See Figure S5 for binding analysis after SGF and SIF digest for other IL-23R minibinder variants. (C) A single oral 20 mg/kg dose of 23R-72 or 23R-91 was administered in healthy rats, formulated in either PBS or GI-protective vehicle (GPV), and minibinder concentration measured in serum and target tissues 6 hours after dosing. Mean ± SD, n=2 technical replicates in analysis, N=5 animals per group. Samples falling below the limit of detection (BLOD) were assigned a value of 0. (D) A single oral 140 mg/kg dose of 23R-91 was administered in healthy rats, and serum concentration of minibinder measured at the indicated time points. Mean ± SD, n=2 technical replicates in analysis, N=6 animals per group. All samples at the 6 hour time point (*) were BLOD. See Table S5 for values and Figure S7 for ELISA standard curves.

Figure 5.. The crystal structure of 23R-91 is very close to the design model.

Both chains in the crystal structure asymmetric unit were aligned via Cɑ atoms to the computational model of 23R-91, with 0.7 Å (chain A) and 0.4 Å (chain B) RMSD. The core binding interface, including native hotspot W3, is indicated with a dashed box. See Table S4 for data collection and refinement stats.

Figure 6.. IL-23R and IL-17 minibinders block cell signaling in primary cells and organoids derived from human skin cells.

(A) Cell suspensions were prepared from the colon, mLN, and spleen of healthy rats, then stimulated with anti-CD3 and rIL-23 (1 μg/mL for colon, 10 ng/mL for mLN and spleen) with or without minibinder 23R-91 (100 nM). After 24 hours incubation, IL-17A was measured in culture supernatants with ELISA. Anti-CD3 only treatment served as a control showing the extent of IL-23-independent IL-17A production, which is not expected to be inhibited by 23R-91 (n=3 replicates per stimulation condition per two independent experiments). (B) IL-23R minibinders block IL-23 signaling in primary human CD4+ T cells with low nanomolar IC50s. Cells were stimulated with recombinant IL-23 with or without a titration of each minibinder for 20 minutes, and stained for CD4 and phosphorylated STAT3. Mean fluorescence intensity (MFI) of pSTAT3 in CD4+ cells was measured by flow cytometry. Mean values ± SD are shown, n=3. (C) Human epithelial organoids were treated with IL-17A (15 nM) with or without minibinder 17–51 (75 nM) and analyzed by qPCR for downstream markers CCL20, CXCL8 (IL-8), and S100A7. Gene expression data were normalized to housekeeping gene HPRT1. Fold change was calculated relative to an untreated control group and is presented as a percent of the response seen with IL-17A-only treatment. Three organoids per stimulation condition were pooled for qPCR analysis in triplicate (n=3) in each of two independent experiments with unique donors (N=2).

Figure 7.. Evaluation of 23R-91 in the NSG-IBD humanized mouse model of colitis.

(A) NSG-IBD study design and schematic. N≥5 animals per group in at least 2 independent studies per condition. (B) On the last day of the study, each animal was assigned a clinical score of overall health. At sacrifice, the colon was dissected and assigned a macroscopic score of inflammation, then prepared for histology to assess microscopic features of inflammation and fibrosis. Scores are plotted as mean ± SD. Treatment groups were compared to challenged control using non-parametric Wilcoxon matched-pair tests (ns = no significance, *P <0.033, **P<0.002, 95% confidence interval). See Table S6 for scoring matrices.