Study design

This was a single-centre, randomized, double-blind, placebo-controlled trial including 38 healthy volunteers aged between 18 and 45 years, conducted at the Centre for Human Drug Research, Leiden, The Netherlands between June and October 2022. Recruitment of participants took place between 27 May and 6 July 2022. All participants gave written informed consent according to the Declaration of Helsinki recommendations prior to any study-related activity. The study was approved by the Independent Ethics Committee of the Foundation ‘Evaluation of Ethics in Biomedical Research’ (Stichting Beoordeling Ethiek Biomedisch Onderzoek), Assen, The Netherlands, was performed according to Good Clinical Practice (GCP) and was registered in the clinicaltrials.gov register under number NCT05682222. All participants underwent medical screening including medical history, physical examination, 12-lead electrocardiography and safety chemistry and haematology blood sampling. The key exclusion criteria were an active or recurrent infection, gastrointestinal tract disease, previous exposure to KLH or diagnosis with psoriasis or eczema. A complete list of the inclusion and exclusion criteria can be found in the Supplementary material.

EDP1815

EDP1815 and placebo capsules were provided by Evelo Biosciences Inc. (Cambridge, MA, USA) and produced by Cambrex (East Rutherford, NJ, USA). Capsules contained lyophilized, thus non-living, cells of a specific bacterial strain of P. Histicola. The selected dose of 8 × 1010 cells per day was well-tolerated, safe and showed clinical efficacy in a prior phase 2 study [16]. The duration of treatment (i.e. 60 days) was based on providing sufficient inhibition of the KLH challenge by already starting treatment 7 days before the first KLH immunization. The two enteric coatings (ECs), EC1 and EC2, had different polymer coating levels, with approximately 58 mg dry-weight enteric polymer coating for EC1, and 14 mg dry-weight enteric polymer coating for EC2.

Treatments and randomization

Participants were studied in 2 cohorts of 18 participants. In each cohort, participants were randomized 2:1 to receive active or placebo treatment, respectively. Placebo participants of both cohorts were pooled in the analysis, resulting in a 1:1 randomization. The randomization code was generated by a study-independent statistician and was made available for data analysis after study completion and database lock. EDP1815 capsules and placebo capsules were identical in appearance and packaging, and were distributed according to the randomization numbers, ensuring concealment of treatment allocation. A schematic overview of the study timeline is shown in Fig. 1. Participants started EDP1815 or placebo treatment (Cohort 1: EC1, Cohort 2: EC2) at day 1 and took one capsule per day for 60 days. Treatment compliance of EDP1815 was measured by paper diaries that were filled in by participants and by counting the remaining capsules that participants handed in.

Fig. 1

Schematic study overview. KLH, keyhole limpet haemocyanin; LSCI, laser speckle contrast imaging

For the KLH challenge, participants received intramuscular KLH immunizations containing 0.1 mg Immucothel® adsorbed into Alhydrogel® containing 1.32 mg Al(OH)3 on days 8, 22 and 36, and received an intradermal skin challenge injection containing 0.001 mg Immucothel® in 0.1 mL NaCl in the arm at day 57. For the imiquimod challenge, participants received topical applications of 100 mg Aldara®, each containing 5 mg imiquimod, under occlusion by a 12-mm Finn chamber (Bipharma, Almere, The Netherlands) at 3 different areas on the back, starting at day 57. There was one area with 24 h of imiquimod exposure (1 application), one area with 48 h of imiquimod exposure (2 applications) and one area with 72 h of imiquimod exposure (3 applications). To ensure sufficient imiquimod delivery through the skin barrier, tape stripping of the skin was conducted before the first application of imiquimod, as described previously [20].

Pharmacodynamic outcomes — imaging-based endpoints

Skin responses were quantified by measuring cutaneous blood perfusion and erythema at 4, 24, 48 and 72 h after the intradermal KLH challenge, and at the same timepoints (except for 4 h) during the topical imiquimod challenge. Cutaneous blood perfusion was measured by laser speckle contrast imaging (LSCI) (PeriCam PSI System, Perimed AB, Järfälla, Sweden), and erythema was measured by multispectral imaging (Antera 3D®, Miravex, Dublin, Ireland), as described earlier [17]. Cutaneous blood perfusion, i.e. basal flow, and homogeneity of cutaneous blood perfusion, i.e. flare, were expressed in arbitrary units (AUs). Erythema was measured using the CIELab a* Antera 3D® software modalities. The CIELab a* value expressed colour as a numerical value on a green-red colour scale, also measured in AUs.

Pharmacodynamic outcomes — humoral immune response to KLH

The specific B-cell response to KLH immunization was measured by anti-KLH IgM and IgG serum titres. Serum samples were obtained by venapuncture in serum clot activator tubes (Vacutainer®) at days 1, 22, 36 and 57, centrifuged at 2000g for 10 min at a temperature of 2–8 °C and aliquoted. Aliquots were stored at a temperature of <−40 °C until shipment and analysis. Antibody levels were measured by quantitative enzyme-linked immunosorbent assay (ELISA) by Ardena Bioanalytical Laboratory (Assen, The Netherlands) and were expressed as relative ratios to the mean optical density of baseline samples.

Pharmacodynamic outcomes — cells and cytokines in blister fluid on imiquimod-treated skin

On the imiquimod-treated skin, suction blisters were induced as described previously [24] at baseline and after respectively 24, 48 and 72 h of imiquimod application. Blister fluid (including blister cells) was collected in a V-bottom plate containing 50 μL 3% sodium citrate (Sigma–Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS; Gibco) and kept on ice. Within 1 h after fluid collection, the fluid was centrifuged to separate the supernatant from the cells in the blister. After centrifuging, the supernatant was collected, and the remaining cell pellet was resuspended in FACS buffer. The cells were stained for flow cytometry analysis with the antibodies listed in Table S1. After staining, the cells were washed and analysed with a MACSQuant 16 analyser (Miltenyi Biotec). The gating strategy of the blister cells is shown in Fig. S1. All flow cytometry data was analysed using absolute cell numbers acquired after staining the cells.

The collected supernatant was weighed to calculate the total amount of fluid per blister and frozen at −80 °C. Cytokine concentrations in the supernatant (tumour necrosis factor [TNF], interferon [IFN]-α, IFN-γ, interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-33 and CXC-motif chemokine ligand 10 [CXCL-10]) were quantified by an immunoassay using Meso Scale Discovery Vplex-2 method (Rockville, MD, USA) by Ardena Bioanalytical Laboratory (Assen, The Netherlands).

Outcome measures

The primary outcome of the study was basal flow 24 h after the intradermal KLH skin challenge. Other pharmacodynamic outcomes of the study were the other imaging outcomes of the KLH and imiquimod challenge as described above, specific B-cell response to KLH immunizations measured by anti-KLH IgM and IgG levels, and influx of cells and cytokines in blister fluid during the imiquimod challenge. Safety and tolerability were monitored by physical examination, assessment of vital signs, laboratory parameters (i.e. full blood count, biochemistry and urine analysis) and ECG data from 12-lead ECGs at regular intervals. Participants were monitored continuously for adverse events (AEs).

Statistics

The sample size was based on the primary endpoint, basal flow (measured by LSCI) 24 h after intradermal KLH skin challenge that showed a standard deviation of 10.3 AU in previous studies. It was calculated that a sample size of 12 in each group would result in a power of 0.80 to detect a difference in means of 12.3 AU, using a two-sample t-test with a 0.05 two-sided significance level. Continuous values of baseline characteristics were summarized by mean and standard deviation; qualitative baseline characteristics by counts and percentages. To detect significant treatment effects, all repeated measured pharmacodynamic (PD) parameters were analysed by a mixed model analysis of covariance (ANCOVA) with treatment, time and treatment by time as fixed factors, the participant as random factor and the (average) baseline measurement as covariate. For all outcome measures, treatment effects were determined for three contrasts: EDP1815-EC1 vs placebo, EDP1815-EC2 vs placebo and EDP1815 overall vs placebo, with each contrast having a separate p-value. Anti-KLH antibodies were analysed without baseline measurement as covariate. Cytokine concentrations in blister fluid were corrected for the volume of each blister and the dilution with 50 μL of PBS sodium citrate. The treatment effects (EDP1815-EC1 vs placebo, EDP1815-EC2 vs placebo, and EDP1815 overall vs placebo) were reported with the estimated difference and the 95% confidence interval, the least square mean estimates (LSM) and the p-values. For PD values below the limit of quantification, a value of half the lower limit of quantification was used. Missing data or assessments were not imputed. p-values < 0.05 were considered statistically significant. All statistical analyses were performed using SAS for Windows version 9.4 (SAS Institute, Inc., Cary, NC, USA). All figures were created using GraphPad Prism version 9 (GraphPad Software, Boston, MA, USA).