Supplementary information
A Straumann et al.
SUPPLEMENTARY METHODS
Biomarker assessment
Blood biomarkers: Eosinophil cationic protein (ECP), eosinophil derived
neurotoxin (EDN), eotaxin, and TNF- were measured in serum at baseline,
weeks 4, 9, 13, and 21 (pre-infusion on treatment days) using commercial
immunoassays except EDN concentrations, which were measured using a
non-commercial immunoassay.25 These assays were performed using frozen
serum samples and all samples from a single patient were analyzed together.
IL-5 surface receptor  expression on eosinophils was measured by direct
immunofluorescence using PE-labeled anti-IL-5 receptor  mAb (StemCell
Technologies, Grenoble, France) and flow cytometry (FACS Calibur, BD
Biosciences, Basel, Switzerland). Eosinophils were identified within the
granulocyte gate by using FITC-labeled anti-CC chemokine receptor 3 (CCR3; R&D Systems Europe Ltd., Abingdon, UK) and FITC-labeled anti-CD9
mAbs (BD Biosciences). Receptor density was determined by analyzing the
mean channel of fluorescence (mcf). For IL-5 receptor  analysis, anticoagulated blood was analyzed within 10 h of collection.
Oesophageal tissue markers: Oesophageal biopsy tissue sections were
obtained at screening (pre-infusion), week 4, and week 13 (as no patient had
demonstrated a histological remission, there were no biopsies taken after
1
STFU. Tissues were fixed in 4% formaldehyde in phosphate buffer (pH 7.4),
embedded, and sectioned. Four proximal and four distal biopsies were
collected at each time point for each patient and embedded in one paraffin
block. Indirect immunostainings were performed as previously described. 26 27
The following primary antibodies: anti-ECP (EG1, Pharmacia & Upjohn
Diagnostics AB, Uppsala, Sweden), anti-CD3 (DakoCytomation, Glostrup,
Denmark),
anti-tryptase
(DakoCytomation),
anti-IL-5
(Santa
Cruz
Biotechnology, Heidelberg, Germany), anti-TNF-α (Novus Biologicals, Inc,
Littleton, CO, USA), anti-transforming growth factor (TGF)-1 (R&D Systems),
anti-eotaxin-1, anti-eotaxin-2, anti-eotaxin-3 (all R&D Systems), and antitenascin C (Monosan, Uden, The Netherlands). Ten hpfs (area of one hpf
0.0538 mm2) were viewed per slide such that 80 counts were made (10 x 8
specimens) for each patient and biomarker at each time point, and the
numbers of positive cells were derived. If epithelial cells expressed a
biomarker, a grading system was used for semiquantitative quantification
using a 5-point scale from 0 to 4: 0 = no positive cells, 1 = weak staining, 2 =
clear positive staining, 3 = strong positive staining, 4 = very strong positive
staining.
Sections were also stained with an anti-EDN antibody and analyzed by
fluorescence microscopy.28 Here, only the two largest of the four available
biopsies in each block were analyzed. To quantify EDN positive cells
(=eosinophilia), each specimen was graded on a 7-point scale from 0 to 3 (0 =
no eosinophils, 3 = confluent eosinophils). To quantify extracellular EDN
deposition, we also used a similar 7-point scale from 0 to 3 (0 = no deposition,
3 = extensive deposition).
2
All stained slides were evaluated by at least two independent
investigators (SC, PG, HK, GK, HUS), and the results were averaged to
obtain a mean for each patient at each time point.
Pharmacokinetic assessment
Blood samples (2 mL) for determination of mepolizumab concentrations were
collected prior to and 5 min after each infusions on days 0 and 7, and weeks 5
and 9. Additional samples were collected at weeks 4, 13, 21 (STFU), 43
(LTFU), as well as at any early withdrawal visit. Plasma was separated and
frozen until analysis. Mepolizumab concentrations were measured using a
validated analytical time-resolved fluorescent immunoassay method with 50
ng/mL sensitivity. Plasma concentration data were summarized by sampling
times.
Immunogenicity assessment
Serum samples for the measurement of mepolizumab binding and
neutralizing antibodies were collected on days 0 and 7, weeks 4, 5, 9, 13, and
21, and LTFU (week 43) (pre-infusion on treatment days) using an
electrochemiluminescent bridging immunoassay according to recommended
methods.29
3
SUPPLEMENTARY RESULTS AND DISCUSSION
Pharmacokinetics
Plasma concentrations of mepolizumab were measured throughout the study.
The mean peak concentration 5 min after a single 750 mg mepolizumab dose
was approximately 180 g/mL, and, after a second 750 mg dose one week
later, it was 267 g/mL (suppl. table 1). Following administration of 1500 mg
mepolizumab on weeks 5 and 9, the observed mean peak concentrations
were approximately 500 g/mL and 450 g/mL, respectively. Mean plasma
concentrations were always greater than 58 g/mL until week 13. At STFU
(12 weeks after last infusion), mepolizumab concentrations were still
detectable and the mean plasma concentration was 8.4 g/mL (suppl. table
1).
At STFU assessment (12 weeks after last infusion), blood eosinophil
numbers were low in the mepolizumab group (fig 2). At this time, plasma
mepolizumab concentrations ranged from 1.5 g/mL to 21.5 g/mL. At LTFU
assessement
(34
weeks
after
last
infusion),
plasma
mepolizumab
concentrations were <0.2 g/mL and eosinophil counts were at or above
baseline values, except for one patient for whom the plasma mepolizumab
concentration was 0.29 g/mL. A correlative description of plasma
mepolizumab concentrations and blood eosinophil numbers suggests that
plasma mepolizumab levels of about 5 g/mL should be sufficient to keep
blood eosinophil numbers below 200/mm3 in EoE patients (suppl. fig 3).
This
study
allows
some
general
conclusions
regarding
the
pharmacological profile of mepolizumab. Although doubling the dose of
4
mepolizumab to 1500 mg increased the peak plasma mepolizumab
concentrations, neither blood nor oesophageal eosinophil numbers decreased
much more than with the lower dose of 750 mg. Mepolizumab plasma half-life
is approximately 20 days (estimated from the data of this study and additional
unpublished data). Consistent with a long half-life and a mean peak plasma
concentration following the second 1500 mg dose of approximately 450
g/mL, plasma concentrations were approximately 5 g/mL or greater at 12
weeks after the last infusion. Blood eosinophil numbers were below 200 per
mm3 during this time. This is likely due to the fact that plasma mepolizumab
concentrations of 5 g/mL were sufficient to keep eosinophil numbers within
the normal range. However, it should be noted that levels of ECP in blood
slightly increased at week 21 (12 weeks after the last infusion) (fig 6), pointing
to the possibility that IL-5 was less efficiently neutralized at this time point.
These results suggest that it might be sufficient to administer 750 or 1500 mg
doses of mepolizumab at 8-12 week intervals in patients receiving long-term
treatment.
Immunogenicity
Sera from two patients treated with mepolizumab were tested positive for
mepolizumab binding antibodies on at least one time point. One patient
(patient 7) was positive with the lowest titer possible for detection 7 days after
the first application of mepolizumab. At later time points, we obtained no
longer evidence for mepolizumab binding antibodies in this patient and the
eosinophil levels in blood remained suppressed through to the STFU
assessment. No adverse effect was reported by this patient. The second
5
patient (patient 8) developed clearly detectable anti-mepolizumab antibodies
after he received all four infusions. The antibodies were detected both during
EW and LTFU (week 43) visits. The patient reported mild fatigue following the
last mepolizumab infusion. The eosinophils remained suppressed at least until
the EW visit, suggesting that the anti-mepolizumab immune response had no
influence on the pharmacological effect of mepolizumab at this time point. The
relationship
between
antibody
production,
mepolizumab
plasma
concentrations, and eosinophil numbers in these two patients is shown in
suppl. table 2.
SUPPLEMENTARY TABLES
Suppl. table 1
Mepolizumab plasma concentrations
Mean (SD)
Day
Dose
Week
range
(mg)
n
(g/mL)
CV (%)
Day 1 (pre-dose)*
1
5
0
0
Day 1 (post-dose)*
1
750
5
181.46 (84.41)
47
Week 1 (pre-dose)
6-7
5
65.54 (27.98)
43
Week 1 (post-dose)
6-7
750
5
266.56 (35.46)
13
Week 4
27-28
5
89.37 (26.06)
29
Week 5 (pre-dose)
34-36
5
57.67 (24.23)
42
Week 5 (post-dose)
34-36
1500
5
502.43 (45.34)
9
Week 9 (pre-dose)
62-63
5
103.74 (36.63)
35
Week 9 (post-dose)
62-63
1500
5
446.45 (182.11)
41
Week 13
90-91
5
116.90 (32.92)
28
Week 21 (STFU)
124-205
5
8.38 (7.86)
94
Week 43 (LTFU)
293-308
5
0.164 (0.088)
54
*Pre-dose is just prior to mepolizumab infusion and post-dose is
approximately 5 min following end of mepolizumab infusion
Abbreviations: STFU, short-term follow-up; LTFU, long-term follow-up;
CV, coefficient of variability
6
Suppl. table 2
Comparison of anti-mepolizumab antibody levels,
mepolizumab plasma concentrations and blood eosinophil counts in antimepolizumab antibody positive patients
Mepolizumab
serum
Actual
Binding
Binding
study
antibody antibody concentration
Patient day
Visit*
assay
(Titre)
(g/mL)
7
1
Baseline
Negative N/A
0
8
Week 1
Positive
2
41.2727
29
Week 4
Negative N/A
93.4086
36
Week 5
Negative N/A
41.6566
64
Week 9
Negative N/A
39.5815
92
Week 13
Negative N/A
67.0014
176
Week 21
Negative N/A
1.3674
295
Week 43
Negative N/A
n.d.
8
1
Baseline
Negative N/A
0
7
Week 1
Negative N/A
32.7390
28
Week 4
Negative N/A
43.4161
35
Week 5
Negative N/A
22.9542
63
Week 9
Negative N/A
107.570.5
91
Week 13
Negative N/A
104.2563
182
Week 21
Negative N/A
5.2969
223
EW
Positive
16
n.d.
300
Week 43
Positive
256
0.1584
*All sampling pre-infusion on treatment days
Abbreviations: EW, early withdrawal; n.d., not done.
Blood
eosinophils
(GI/L)
0.54
0.06
0.10
0.04
0.05
0.09
0.12
1.03
0.56
0.14
0.04
0.06
0.05
0.06
0.06
0.11
0.34
7
SUPPLEMENTARY FIGURES
Suppl. figure 1
Disposition of the patients. STFU, short-term follow-
up (12 weeks after the last infusion). LTFU, long-term follow-up (34
weeks after the last infusion).
Screened for eligibility (n=13)
Screen failures
Eos <20 per hpf (n=2)
Randomised (n=11)
Mepolizumab (n=5)
Completed STFU (n=5)
Placebo (n=6)
Completed STFU (n=6)
Withdrawn
Worsening EoE (n=1)
Completed LTFU (n=4)
Withdrawn
Worsening EoE (n=2)
Completed LTFU (n=4)
Suppl. Figure 1
8
Suppl. figure 2
Identification of esophageal infiltrating cells by
immunofluorescence analysis. Representative original results of the
identification of eosinophils (ECP positive cells), T cells (CD3 positive
cells), and mast cells (tryptase positive cells) before and after
mepolizumab and placebo treatment, respectively, are shown (x400).
Quantitative analysis of these experiments is shown in the table 3.
Placebo
Before
Mepolizumab
After
Before
After
ECP
CD3
Tryptase
Suppl. Figure 2
9
Suppl. figure 3
Relationship
between
eosinophil
numbers
and
mepolizumab concentrations in blood.
Eosinophil Count (GI/L)
1.2
1
0.8
Placebo
0.6
Mepolizumab
0.4
0.2
0
0
50
100
150
200
Mepolizumab Concentration (g/mL)
Suppl. Figure 3
10