Research ArticleCORONAVIRUS

Inhibition of Bruton tyrosine kinase in patients with severe COVID-19

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Science Immunology  05 Jun 2020:
Vol. 5, Issue 48, eabd0110
DOI: 10.1126/sciimmunol.abd0110
  • Fig. 1 Model of BTK-dependent hyperinflammation in severe COVID-19.

    Binding of SARS-CoV-2 to ACE2 on respiratory epithelia initiates infection. Hypothetically, macrophages may participate in the COVID-19 inflammatory response by phagocytic uptake of viral particles or cellular debris containing viral ssRNA. ssRNA can bind to TLR7 and TLR8, or to TLR3 using double-stranded regions in its secondary structure, thereby recruiting and activating BTK (51, 52). Downstream of TLR engagement, BTK-dependent NF-κB activation results in the production of proinflammatory cytokines and chemokines (53), a cytokine storm that could increase the recruitment of monocytes/macrophages and neutrophils during the late phase of severe COVID-19 infection. BTK inhibitors such as acalabrutinib block TLR-dependent NF-κB activation in macrophages (20, 21), thereby dampening the production of proinflammatory mediators, as occurs in an influenza-induced lung injury model (27). During severe COVID-19, the heightened levels of IL-1β in several patients with COVID-19 (11, 12) indicate the formation of an NLRP3 inflammasome that converts pro–IL-1β to mature IL-1β (54). BTK binds to and phosphorylates NLRP3, thereby promoting its oligomerization and assembly into an inflammasome (2426). BTK inhibitors such as acalabrutinib inhibit inflammasome-mediated production of IL-1β, as observed in a model of influenza-induced lung injury (27). ACE2, angiotensin-converting enzyme 2; IL-12R, IL-12 receptor; CCL2, C-C motif chemokine ligand 2; CXCR2, C-X-C motif chemokine receptor 2; IFNG, interferon gamma G; NLRP3, NLR family pyrin domain containing 3; P, phosphate.

  • Fig. 2 Clinical course and changes in inflammatory markers during acalabrutinib treatment in patients treated before intubation.

    Shown are measures of oxygen uptake requirement SpO2/FiO2 [% blood oxygen saturation (SpO2)/fraction of delivered oxygen (FiO2)], a ratio that accounts for both oxygen delivery and uptake (theoretical maximum 476 for 100% oxygen saturation on room air). Also shown are measures of inflammation (CRP, mg/dl) and ALC (cells/μl) at all available time points before and after acalabrutinib treatment, which was started on day 1 (dashed line). Notable clinical parameters are shown as indicated (extubation, breathing on room air, transfer to rehabilitation, hospital discharge, and death). The duration of mechanical ventilation (Vent.) is indicated.

  • Fig. 3 Clinical course and changes in inflammatory markers during acalabrutinib treatment in patients treated while on mechanical ventilation.

    See legend in Fig. 2.

  • Fig. 4 Associations between measures of pulmonary function and inflammation after acalabrutinib treatment.

    (A) Plots of oxygen uptake efficiency (SpO2/FiO2), CRP, and ALC levels versus days of acalabrutinib treatment for all patients at all time points. Patients in the supplemental oxygen and mechanical ventilation cohorts are indicated in red and blue, respectively. The trend lines shown represent the regression from a linear mixed-effect model blocked by patient. The reported P values test the null hypothesis that the trend line has zero slope. (B) Plots of oxygen uptake efficiency (SpO2/FiO2) versus either CRP or ALC. Trend lines and P values as above.

  • Fig. 5 BTK activation and IL-6 production in COVID-19.

    (A) Left: Histograms of BTK phosphorylation in CD14+ blood monocytes from three patients with severe COVID-19 (A, B, and C; table S10) and four healthy volunteers, as indicated. Right: Summary data showing significant increase in MFI of phosphorylated BTK (residue Y223) in CD14+ monocytes from three patients with COVID-19 compared with five healthy volunteers, with no evident BTK phosphorylation in CD19+ B cells. Total BTK levels in blood monocytes shown in the far right panel were comparable in three patients with COVID-19 and five healthy volunteers. (B) Left: Representative contour plots of intracellular IL-6 production in CD14+ monocytes from a patient with severe COVID-19 (patient C; table S10) and a healthy volunteer, either as unstimulated ex vivo cells or after R848 (10 μM) stimulation, as indicated. Right: Summary data showing significant increase in the percent of IL-6+ CD14+ monocytes from four patients with COVID-19 (A, B, C, and D; table S10) compared with five healthy volunteers, before and after R848 stimulation, with no evident IL-6 production by CD19+ B cells. (C) Plot of blood IL-6 concentrations (pg/ml) on a log scale versus days of acalabrutinib treatment for patients in whom there were at least two IL-6 measurements during the plotted time course. Patients in the supplemental oxygen (n = 5) and mechanical ventilation (n = 3) cohorts are indicated in red and blue, respectively. The trend line shown represents the regression from a linear mixed-effect model blocked by patient for the combined cohorts due to limited data in each group. The reported P value tests the null hypothesis that the trend line has zero slope. All quantitative data in (A) and (B) represent means ± SEM.

  • Table 1 Characteristics of the patients.

    Obesity is defined as body mass index ≥ 30 kg/m2; morbid obesity is defined as body mass index ≥ 40 kg/m2.

    All patients (N = 19)Supplemental oxygen (N = 11)Mechanical ventilation (N = 8)
    Male sex – no. of patients (%)13 (68%)6 (55%)7 (88%)
    Age – median (range) (years)61 (45–84)62 (48–84)61 (45–77)
    Ethnicity
      White9 (47%)6 (55%)3 (38%)
      Hispanic5 (26%)2 (18%)3 (38%)
      Black3 (16%)2 (18%)1 (13%)
      Asian1 (5%)01 (13%)
      Middle Eastern1 (5%)1 (9%)0
    Days of symptoms – median (range)7 (2–21)9 (2–21)7 (3–21)
    Days of treatment – median (range)10 (4–14)10 (4–14)10 (6–14)
    Required intubation after
    treatment – no. of patients (%)
    2 (18%)
    Days on ventilator before
    treatment – median (range)
    1.5 (1 to 22)
    Extubated after treatment – no. of
    patients (%)
    4 (50%)
    Comorbid conditions
    Hypertension16 (84%)9 (82%)7 (88%)
    Obesity13 (68%)8 (73%)5 (63%)
      Morbid obesity5 (26%)3 (27%)2 (25%)
    Diabetes mellitus7 (37%)3 (27%)4 (50%)
    Obstructive sleep apnea3 (16%)2 (18%)1 (13%)
    Chronic kidney disease2 (11%)1 (9%)1 (13%)
    Coronary artery disease1 (5%)1 (9%)0
    Chronic obstructive lung disease1 (5%)1 (9%)0
    Asthma1 (5%)1 (9%)0
    Sarcoidosis1 (5%)01 (13%)
    Rheumatoid arthritis1 (5%)1 (9%)0
    Chronic lymphocytic leukemia1 (5%)1 (9%)0
    Prostate cancer1 (5%)1 (9%)0
    Signs and symptoms
    Cough15 (79%)8 (73%)7 (88%)
    Dyspnea14 (74%)10 (91%)4 (50%)
    Fever12 (63%7 (64%)5 (63%)
    Myalgias/muscle weakness6 (32%)3 (27%)3 (38%)
    Diarrhea2 (11%)02 (25%)
    Vomiting1 (5%)01 (13%)
    Sore throat1 (5%)1 (9%)0
    Loss of taste1 (5%)1 (9%)0
    Laboratory values
    ALC (cells/μl)
      ≤100015 (83%)8 (80%)7 (88%)
      >10003 (17%)2 (20%)1 (12%)
      *Not applicable11
    CRP (mg/dl)
    ≥1015 (79%)7 (64%)8 (100%)
      >3 and <104 (21%)4 (36%)
      <30
    Serum ferritin (ng/ml)
      ≥50016 (84%)8 (73%)8 (100%)
      <5003 (16%)3 (27%)
    Fibrinogen (mg/dl)
      ≥40010 (100%)6 (100%)4 (100%)
      <4000
      Unknown954
    D-dimer (mcg/ml)
      ≥0.515 (88%)8 (80%)7 (100%)
      <0.52 (12%)2 (20%)
      Unknown211
    IL-6 levels (pg/ml)
      ≥159 (100%)6 (100%)3 (100%)
      <150
      Unknown1055

    *One patient had preexisting chronic lymphocytic leukemia making the lymphocyte count uninterpretable.

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/5/48/eabd0110/DC1

      Supplementary Methods

      Fig. S1. Gating strategy for flow cytometric analysis of phosphorylated and total BTK.

      Fig. S2. Gating strategy for flow cytometric analysis of IL-6.

      Table S1. Treatment centers.

      Table S2. Clinical course of supplemental oxygen cohort.

      Table S3. Clinical course of mechanical ventilation cohort.

      Table S4. Laboratory tests for inflammatory markers during acalabrutinib treatment in supplemental oxygen cohort.

      Table S5. Laboratory tests for inflammatory markers during acalabrutinib treatment in mechanical ventilation cohort.

      Table S6. Other laboratory tests during acalabrutinib treatment in supplemental oxygen cohort.

      Table S7. Other laboratory tests during acalabrutinib treatment in mechanical ventilation cohort.

      Table S8. Statistical analysis of laboratory changes related to oxygenation status.

      Table S9. Adverse events of special interest during treatment with acalabrutinib.

      Table S10. Characteristics of patients with COVID-19 who underwent flow cytometry–based immunological analyses.

      Table S11. Raw data file (Excel spreadsheet).

    • Supplementary Materials

      This PDF file includes:

      • Preparation of Acalabrutinib For Enteric Feeding Tube Administration
      • Questions and Answers Regarding Acalabrutinib For Enteric Feeding Tube Administration
      • Table S1. Treatment centers.
      • Table S2. Clinical course of supplemental oxygen cohort.
      • Table S3. Clinical course of mechanical ventilation cohort.
      • Table S4. Inflammatory laboratory tests during acalabrutinib in supplemental oxygen cohort.
      • Table S5. Inflammatory laboratory test during acalabrutinib in mechanical ventilation cohort.
      • Table S6. Other laboratory tests during acalabrutinib in supplemental oxygen cohort.
      • Table S7. Other laboratory tests during acalabrutinib in mechanical ventilation cohort.
      • Table S8. Statistical analysis of laboratory changes related to oxygenation status.
      • Table S9. Adverse events of special interest during treatment with acalabrutinib.
      • Table S10. Characteristics of COVID-19 patients who underwent flow cytometry-based immunological analyses.
      • Fig. S1. Gating strategy for flow cytometric analysis of phosphorylated and total BTK.
      • Fig. S2. Gating strategy for flow cytometric analysis of IL-6.

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      Other Supplementary Material for this manuscript includes the following:

      • Table S11. Raw data file (Excel spreadsheet).

      Files in this Data Supplement:

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