Research ArticleAutoimmunity

Dynamics of circulating TNF during adalimumab treatment using a drug-tolerant TNF assay

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Science Translational Medicine  30 Jan 2019:
Vol. 11, Issue 477, eaat3356
DOI: 10.1126/scitranslmed.aat3356
  • Fig. 1 Development of a drug-tolerant competition ELISA.

    (A) Schematic overview of the drug-tolerant competition ELISA. Both free TNF and TNF-adalimumab complexes in serum are bound to an anti-TNF–coating antibody. An excess of a biotinylated high-affinity adalimumab mutant antibody is added, which will result in the displacement of adalimumab from TNF, allowing efficient detection of TNF. Measurement of free TNF and TNF-adalimumab complexes in the drug-tolerant competition ELISA (B) and in a conventional TNF ELISA (C). Shown is a representative titration of free TNF, preincubated in absence or presence of adalimumab (5 μg/ml) of at least four independent experiments.

  • Fig. 2 Characterization of TNF-adalimumab complexes.

    (A) Correlation between TNF measured in sera, sent in to Sanquin Diagnostic Services, using the competition ELISA and complex ELISA (Pearson r = 0.96, P < 0.0001; n = 36). Samples spiked with free TNF (B) and TNF preincubated with adalimumab (C) in buffer containing IVIg and albumin were fractionated with HP-SEC (gray line). The two peaks around 12.5 and 14 ml represent IgG and albumin, respectively. TNF was measured in collected fractions using the drug-tolerant competition ELISA (black line). Representatives of at least two independent experiments are shown. (D) Characterization of TNF-adalimumab complexes in serum derived from adalimumab-treated patients (n = 4). A representative example of a patient is shown.

  • Fig. 3 Quantification of TNF in adalimumab-treated patients with RA.

    (A) TNF serum concentrations were determined at baseline (week 0) and after 4, 16, 28, 40, 52, 78, and 104 weeks of adalimumab treatment in 193 patients with RA using the drug-tolerant competition ELISA. Each dot represents mean TNF concentration of a duplicate measurement in an individual patient; black lines show median (IQR). (B) TNF activity in a selection of sera from patients with RA (n = 70) during adalimumab treatment. WEHI-164 cells were incubated with 1:20 diluted patient serum (diluted in assay medium) in triplicate. Dotted line shows the optical density (OD) of a noninhibited reference sample. (C) Representative examples of patients with increased TNF concentrations during the first phase of treatment and stabilized TNF concentrations over time. (D) Representative examples of patients with diminished TNF concentrations over time. (A, C, and D) Dotted lines represent cutoff of TNF (5 pg/ml).

  • Fig. 4 Quantification of TNF in patients with RA after adalimumab treatment prolongation or treatment discontinuation.

    (A) Longitudinal adalimumab concentration or (B) TNF concentration in 21 patients with RA before (week 0) and 12 and 28 weeks after treatment prolongation. (C) Longitudinal adalimumab serum concentration or (D) TNF concentration in 11 patients with RA before (week 0) and 12 and 24 weeks after adalimumab treatment discontinuation. Colored lines in (C) correspond to patients with similar colored lines in (D). (B and D) Dotted lines indicate cutoff of TNF (5 pg/ml). (C) Dotted line indicates lower limit of quantification (LLOQ) of adalimumab (0.01 μg/ml).

  • Fig. 5 TNF concentrations at week 4 in relation with clinical response.

    (A) Correlation between TNF concentrations at week 4 and disease activity, according to SDAI, at week 52. (Spearman’s ρ = −0.21, P < 0.005; n = 168). Gray line indicates log-log linear fit, weight by 1/Y2. (B) TNF concentrations were stratified by ADA detection during 52 weeks of follow-up. Each dot represents mean TNF concentration of a duplicate measurement in an individual patient; black lines show median (IQR); patients were only included in the analysis if (free) adalimumab concentrations exceeded 0.1 μg/ml. ****P < 0.0001, Mann-Whitney U test. (A and B) Dotted lines indicate cutoff of TNF (5 pg/ml). (C) ROC analysis of TNF concentrations at week 4 (black line). To predict ADA formation versus no ADA formation after 52 weeks of adalimumab treatment, an AUC of 0.79 was found, and a cutoff value of TNF (11 pg/ml) yielded 51% sensitivity and 95% specificity. Inclusion of baseline MTX usage in the model resulted in an AUC of 0.81 (gray line).

  • Fig. 6 Quantification of TNF in healthy volunteers.

    (A) TNF serum concentrations were determined before (day 0), and frequently after one dose of an adalimumab biosimilar in 30 healthy volunteers, using the drug-tolerant competition ELISA. TNF concentrations were stratified by volunteers in whom no ADAs were detected (gray triangles; n = 18) and volunteers with detectable ADAs over 42 days of follow-up (orange dots; n = 12). Each symbol represents mean TNF concentration of a duplicate measurement in an individual volunteer; gray and orange lines show median TNF for ADA negative and ADA positive, respectively. (B) The dynamics in TNF (black dots), ADA titer (gray triangles; both left y axis) and adalimumab concentration (orange squares; right y axis) in a volunteer who became ADA positive. A representative example of one healthy volunteer is shown. Black and gray dotted lines indicate a cutoff of TNF (5 pg/ml) or a limit of detection (LOD) of anti-adalimumab antibodies [12 arbitrary units (AU)/ml], respectively.

  • Table 1 Demographics, previous and concomitant therapies, and disease status at baseline.

    SDAI, simplified disease activity index; no., number; BMI, body mass index; MTX, methotrexate; ACPA, anticitrullinated protein antibody; IgM-RF, immunoglobulin M rheumatoid factor; DAS28, 28-joint disease activity score; ESR, erythrocyte sedimentation rate; HAQ, health assessment questionnaire.

    Patients (n = 193)
    Demographics
    Age, means ± SD (years)54 ± 11
    Female, no. (%)148 (77)
    BMI, means ± SD25.7 ± 5.1
    DMARD therapy
    Previous DMARDs, median (IQR)3 (2–4)
    MTX use, no. (%)149 (77)
    MTX dose, median (IQR) (mg/week)25 (15–25)
    Prednisone use, no. (%)60 (31)
    Prednisone dose, median (IQR) (mg/day)7 (5–10)
    Disease status
    Disease duration, median (IQR) (years)8 (3–17)
    ACPA positive, no. (%)138 (72)
    IgM-RF positive, no. (%)135 (70)
    Erosive, no. (%)143 (74)
    DAS28, means ± SD5.0 ± 1.3
    SDAI, means ± SD21 (14–27)
    ESR, medians (IQR) (mm/hour)21 (9–42)
    CRP, median (IQR) (mg/liter)11 (4–23)
    HAQ, median (IQR)1.1 (0.8–1.6)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/477/eaat3356/DC1

    Fig. S1. Analysis of antigen binding.

    Fig. S2. TNF concentrations measured with the drug-tolerant competition ELISA.

    Fig. S3. Development of a TNF-adalimumab complex ELISA.

    Fig. S4. Adalimumab-TNF complexes.

    Fig. S5. Correlation between TNF and adalimumab concentrations.

    Fig. S6. TNF concentrations at week 52 and at steady state in relation with clinical response.

    Fig. S7. Validation of the relation between TNF concentrations at week 4 and clinical response.

    Fig. S8. Different adalimumab cutoff concentrations did not affect the relation between TNF concentrations at week 4 and clinical response.

    Fig. S9. TNF concentrations at week 4 in relation with baseline methotrexate use.

    Fig. S10. Adalimumab concentrations and ADAs in healthy volunteers.

    Table S1. Demographics of validation cohort.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Analysis of antigen binding.
    • Fig. S2. TNF concentrations measured with the drug-tolerant competition ELISA.
    • Fig. S3. Development of a TNF-adalimumab complex ELISA.
    • Fig. S4. Adalimumab-TNF complexes.
    • Fig. S5. Correlation between TNF and adalimumab concentrations.
    • Fig. S6. TNF concentrations at week 52 and at steady state in relation with clinical response.
    • Fig. S7. Validation of the relation between TNF concentrations at week 4 and clinical response.
    • Fig. S8. Different adalimumab cutoff concentrations did not affect the relation between TNF concentrations at week 4 and clinical response.
    • Fig. S9. TNF concentrations at week 4 in relation with baseline methotrexate use.
    • Fig. S10. Adalimumab concentrations and ADAs in healthy volunteers.
    • Table S1. Demographics of validation cohort.

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    Other Supplementary Material for this manuscript includes the following:

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