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Proof-of-concept clinical trial of etokimab shows a key role for IL-33 in atopic dermatitis pathogenesis

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Science Translational Medicine  23 Oct 2019:
Vol. 11, Issue 515, eaax2945
DOI: 10.1126/scitranslmed.aax2945
  • Fig. 1 Study design.

    (A) Schematic of the study design. Saline was injected on the inside of the left upper arm, and 0.05 μg of HDM was injected on the inside of the right upper arm, 4 days after receiving either placebo or etokimab [300 mg intravenously (IV)]. After 1 hour, suction blister cups were applied to the sites of saline and HDM challenge with a vacuum pressure of 250 mmHg for 60 to 90 min to generate blisters. The blisters were protected overnight, and interstitial fluid and cellular infiltrate were assessed 24 hours after challenge by ELISA or FACS. (B) Schematic of skin blister analysis. The blister/interstitial fluid from all donors was collected and stored at −80°C, and the cellular infiltrate was analyzed by FACS.

  • Fig. 2 Etokimab induces rapid and sustained improvements in disease severity scores and reported outcome measures.

    AD severity scores after administration of placebo on day −7 and etokimab (300 mg IV) on day 1. (A) EASI score relative to day −14 (100%). (B) Percentage of patients reaching EASI50 and EASI75. (C) Changes in absolute EASI score. (D) Changes in absolute SCORAD score. (E) Changes in IGA absolute score. (F) Percentage DLQI score relative to baseline. (G) Percentage 5D itch score relative to baseline. Friedman test with Dunn’s multiple comparison. n = 12, showing means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 Pharmacodynamic effects after administration of placebo on day 1 and etokimab (300 mg IV) on day 8.

    (A) Whole blood from different time points was incubated at 37°C for 16 hours with IL-12 and IL-33 at 30 and 50 ng/ml, respectively. IFNγ concentration in the supernatant was measured by ELISA and normalized to baseline time point (100%). Friedman test with Dunn’s multiple comparison. n = 12, showing mean ± SD. (B) Peripheral blood eosinophil count (109/liter), showing mean ± SD. (C) Correlation between eosinophil percentage and EASI score (Spearman correlation). *P < 0.05, **P < 0.01, ****P < 0.0001.

  • Fig. 4 Skin suction blisters represent a model of sterile inflammation in humans.

    (A) Leukocytes from skin suction blisters of saline and HDM challenges were analyzed for their forward scatter (FSC) and side scatter (SSC) by FACS. One representative result is shown of 12 donors, and overall analysis of the leukocyte numbers was plotted (right). (B) Leukocytes from skin suction blisters of saline and HDM challenges after etokimab administration were analyzed for their FSC and SSC by FACS. One representative result is shown of 12 donors. (C) Five days after placebo or 300-mg etokimab, skin suction blisters were sampled 24 hours after saline or HDM skin challenge. Granulocytes were quantified using FACS and expressed as percentage of total leukocytes. (D) Five days after placebo, skin suction blisters were sampled 24 hours after saline or HDM skin challenge. Neutrophils were quantified using FACS and expressed as percentage of total leucocytes. (E) Overall analysis of the neutrophil percentage of leukocytes from skin suction blisters of saline and HDM challenges, 5 days after placebo or 300 mg of etokimab. n = 12. Lines in the statistics plots represent the means ± SD. Boxes show 25/75 percentiles, and whiskers show range. *P < 0.05, t test. ns, not significant.

  • Fig. 5 Skin interstitial fluid induces neutrophil migration and activation ex vivo.

    (A) Whole-blood cells (2 × 105 cells) from healthy donors were placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing medium or nonautologous blister fluid at 37°C for 1 hour. The cell numbers from the lower wells were calculated, and then the total cells (left) were collected for FACS analysis. One representative result is shown of three experiments (middle), and the overall numbers of migrated neutrophils was determined (right). n = 5. Lines in the statistics plots represent the means ± SD. (B) Neutrophils (1 × 106 cells) were treated with medium or blister fluid for 24 hours. The concentration of elastase in the blister fluids before and after neutrophil culturing was measured by ELISA. n = 5. (C) Images of neutrophils (0.5 × 105 to 1 × 105 cells) were taken 1 hour after culturing with medium or blister fluid. One representative result is shown of 15 experiments from five donors. The cell morphologies were analyzed, and a circularity index for each cell was assigned. 0 is square and 1.0 is perfect circle. The average circularity index per image was calculated, and overall analysis was plotted. *P < 0.05, **P < 0.01, ****P < 0.0001, t test. Boxes show 25/75 percentiles, and whiskers show range.

  • Fig. 6 Etokimab inhibits IL-33–induced neutrophil migration ex vivo.

    (A) Freshly isolated neutrophils from healthy donors were analyzed for ST2 and IL-1RAcP expression by FACS. n = 3 to 6. FMO, fluorescence minus one. (B) Freshly isolated neutrophils were used for Transwell assays. Neutrophils (2 × 105 cells) were placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing IL-33 at different concentrations at 37°C for 1 hour. Data are shown as fold change of random migration (10% FCS in RPMI 1640). n = 6. (C) The concentrations of IL-33 in the culture with or without different concentrations of etokimab were measured by ELISA. IL-33 (50 ng/ml) added is shown as a dashed line. (D) Neutrophils (2 × 105 cells) were placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing IL-33 (50 ng/ml) with isotype control (5 μg/ml) or etokimab (5 μg/ml) at 37°C for 1 hour. Boxes show 25/75 percentiles, and whiskers show range. n = 8. *P < 0.05, one-way ANOVA or nonlinear fit.

  • Fig. 7 IL-33 has a dominant nonredundant upstream role in sterile skin neutrophilic inflammation.

    Freshly isolated neutrophils (2 × 105 cells) were used for Transwell assays. Neutrophils were (A) pretreated (n = 8) or (B) not pretreated (n = 5) with etokimab (5 μg/ml) for 1 hour and placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing combinations of IL-8 (50 ng/ml), isotype control (5 μg/ml), and/or etokimab (5 μg/ml) at 37°C for 1 hour. (C) Neutrophils were not pretreated with etokimab and placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing blister fluid, isotype control (5 μg/ml), or etokimab (5 μg/ml) at 37°C for 1 hour. n = 4. (D) The concentration of IL-8 in the blister fluids before and after etokimab administration was measured by ELISA. n = 4. (E) IL-8 was measured by ELISA after neutrophils (1 × 106 cells) were cultured in blister fluid with isotype control (5 μg/ml) or etokimab (5 μg/ml) for 24 hours. n = 5. (F) Neutrophils were pretreated with etokimab (5 μg/ml) for 1 hour and placed into the upper wells of a Transwell chamber system and allowed to migrate through 5-μm pores toward the lower wells containing blister fluid with isotype control (5 μg/ml) or etokimab (5 μg/ml) at 37°C for 1 hour. Data are shown as fold change of random migration. n = 7. Boxes show 25/75 percentiles, and whiskers show range. *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test or two-way ANOVA.

  • Fig. 8 Etokimab inhibits IL-33–induced neutrophil autocrine expression of CXCR1 and CXCR2.

    (A) Neutrophils (2 × 105 to 5 × 105 cells) were cultured in medium containing isotype control (5 μg/ml) or etokimab (5 μg/ml) at 37°C for 1, 2, and 3 hours. The expression of CXCR1 and CXCR2 was analyzed by FACS. One representative histogram is shown. n = 4 to 5. (B and C) Neutrophils (2 × 105 to 5 × 105 cells) were cultured in medium containing isotype control (5 μg/ml) or different dosage of etokimab (0.625, 1.25, and 5 μg/ml) at 37°C for 2 hours. (B) The expression of CXCR1 was analyzed by FACS. n = 5. (C) Neutrophil migration toward IL-8 (50 ng/ml) was analyzed against the expression of CXCR1. Correlations between migration fold change and CXCR1 mean fluorescence intensity (MFI) expression (R2 = 0.2606, P = 0.0108) are shown. Each dot represents an individual paired fold change experiment. Boxes show 25/75 percentiles, and whiskers show range. *P < 0.05, **P < 0.01, ***P < 0.005, one-way or two-way ANOVA or linear regression.

  • Table 1 Treatment-emergent AEs.

    ConditionPatient, n (%)ConditionPatient, n (%)
    Nervous system disorders4 (33.3)Musculoskeletal and connective
    tissue disorders
    3 (25.0)
    Dizziness1 (8.3)Back pain1 (8.3)
    Headache3 (25.0)Bursitis1 (8.3)
    Migraine1 (8.3)Pain in extremity1 (8.3)
    Paresthesia1 (8.3)Respiratory, thoracic, and
    mediastinal disorders
    2 (16.7)
    Presyncope0Cough1 (8.3)
    Infections and infestations4 (33.3)Nasal congestion1 (8.3)
    Upper respiratory tract infection2 (16.7)Oropharyngeal pain0
    Urinary tract infection2 (16.7)Vascular disorders1 (8.3)
    Skin infection1 (8.3)Hot flush1 (8.3)
    Injury, poisoning, and procedural
    complications
    3 (25.0)Hypertension0
    Accident1 (8.3)Peripheral coldness0
    Arthropod bite1 (8.3)Psychiatric disorders2 (16.7)
    Clavicle fracture1 (8.3)Depression1 (8.3)
    Contusion0Stress1 (8.3)
    General disorders and administration
    site conditions
    3 (25.0)Skin and subcutaneous tissue
    disorders
    2 (16.7)
    Peripheral swelling2 (16.7)Urticaria2 (16.7)
    Chest pain1 (8.3)Ear and labyrinth disorders1 (8.3)
    Fatigue1 (8.3)Tinnitus1 (8.3)
    Infusion site pain0Gastrointestinal disorders1 (8.3)
    Investigations3 (25.0)Dyspepsia1 (8.3)
    Hemoglobin decreased1 (8.3)Nausea1 (8.3)
    Monocyte count decreased1 (8.3)Vomiting1 (8.3)
    Neutrophil count decreased1 (8.3)

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/515/eaax2945/DC1

    Materials and Methods

    Fig. S1. Peripheral eosinophil percentage and EASI score before treatment with etokimab.

    Fig. S2. Skin blister leukocytes.

    Fig. S3. Etokimab binding to IL-33.

    Fig. S4. Neutrophil migration and CXCR1.

    Table S1. Characteristics of the patients.

    Data file S1. Primary data.

    Reference (46)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Peripheral eosinophil percentage and EASI score before treatment with etokimab.
    • Fig. S2. Skin blister leukocytes.
    • Fig. S3. Etokimab binding to IL-33.
    • Fig. S4. Neutrophil migration and CXCR1.
    • Table S1. Characteristics of the patients.
    • Reference (46)

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    Other Supplementary Material for this manuscript includes the following:

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