1
Pharmacokinetics, pharmacodynamics and safety of QGE031
2
(ligelizumab), a novel high affinity anti-IgE antibody, in atopic subjects
3
4
Jonathan P. Arm, Ivan Bottoli, Andrej Skerjanec, David Floch, Andrea Groenewegen,
5
Suzanne Maahs, Charles E. Owen, Ieuan Jones and Philip J. Lowe
6
7
Supplementary Materials
8
9
Methods
10
Preclinical pharmacology
11
Equilibrium constant:
12
Each monoclonal antibody (omalizumab and QGE031) was diluted to 5 μg/mL in 10 mM
13
sodium acetate pH 4.5 and immobilized at a flow rate of 5 μL/min on a CM5 biosensor chip
14
by standard amine coupling with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
15
hydrochloride, N-hydroxy-succinimide and ethanolamine-hydrochloride (Biacore, Uppsala,
16
Sweden). All monoclonal antibodies were tested by surface plasmon resonance on a Biacore
17
3000 optical biosensor (Biacore, Uppsala, Sweden) at 22°C against polyclonal human IgE
18
analytes. Human IgE analytes were injected onto the chip at five concentrations (0.625, 1.25,
19
2.5, 5 and 10 nM) at a flow rate of 30 μL/min for 5 min and allowed to dissociate for 30 min.
20
The chip surface was regenerated at 100 μL/min with 100 μL of 100 mM HCl. The ligand
21
immobilization level was set at 100 RU. Sensorgrams were plotted with Biacore 300 control
22
software (version 4.1).
1
1
2
Inhibition of IgE binding to cell-surface FcεRI and the immobilized α-subunit of FcεRI:
3
A predetermined amount of biotinylated human IgE was mixed with serially diluted
4
monoclonal antibodies (QGE031 and omalizumab) and added to RBL-2H3 (a gift from Dr F.
5
Kricek, Novartis Dermatology, Vienna, Austria) cells expressing the α-subunit of the human
6
FcεRI receptor on their cell surface. After washing, bound IgE was detected using FITC-
7
conjugated streptavidin and mean fluorescence intensity was determined.
8
9
Serially diluted monoclonal antibodies (QGE031 and omalizumab) were loaded onto
an ELISA plate coated with 1 μg/mL recombinant soluble human FcεRIα receptor. IgE
10
(200 ng/mL) was added to the plate and allowed to bind for 1 h at room temperature, thus
11
allowing IgE the opportunity to bind either with the monoclonal antibodies or the receptor.
12
The plates were washed, and IgE bound to the receptor was determined by a horseradish
13
peroxidase-labeled murine anti-human IgE monoclonal antibody, E10-10 (Tanox Inc,
14
Houston, TX, USA).
15
16
Mast cell degranulation and activation assays:
17
Human cord blood-derived mast cells at a concentration of 1 x 105 cells/80 μL in culture
18
medium supplemented with 25 ng/mL recombinant IL-4 were added to wells of 96-well V-
19
bottom plates. Ten μL aliquots of serial dilutions of monoclonal anti-IgE antibodies
20
(omalizumab, QGE031) or an isotype-matched control IgG1 monoclonal antibody were added
21
to the cells, followed by JW8 3-nitro-4-hydroxy-iodophenyl acetic acid (NIP)-specific IgE to
22
a concentration of 1 nM and incubated overnight. Cells were then spun, washed once with
23
200 μL of culture medium and resuspended in 90 μL of the assay medium. Ten μL of the
24
stimulus (100 ng/mL of NIP-BSA conjugated hapten antigen) or 10 μL of assay medium was
2
1
added and the plates were incubated for 2 h at 37°C. The plates were then spun. Cell pellets
2
from three replicates were combined and stained with anti-CD63-PE monoclonal antibody
3
(Immunotech) to determine the expression of CD63 at the cell surface by flow cytometry. β-
4
hexosaminidase release was measured as described previously [1].
5
6
Binding activity of QGE031 across mammalian species:
7
Binding activity of QGE031 against IgE from different mammalian species was investigated
8
using ELISA measured by absorbance at 450 nm for rat (Zymed), cat (Heska), dog (Heska),
9
and human (Biodesign), while QGE031 binding to cynomolgus non-human primate was
10
11
assessed by Biacore 3000.
Immulon II U-bottom 96-well plates were coated with 50 μL/well of 1μg/mL of
12
polyclonal human IgE, mouse IgE, cat IgE, rat IgE and dog IgE overnight at 4°C. Coating
13
solutions were removed and the plate blocked with 3% bovine serum albumin in phosphate
14
buffered saline (200 μL/well) for 1 h at room temperature. After washing the plates five
15
times, 50 μL/well of primary antibodies or control antibody were added and incubated for 1 h
16
at room temperature, followed by a further five washes. Goat anti-human IgG Fc-horseradish
17
peroxidase (1:2000) was added, incubated for 1 h at room temperature and the plates were
18
washed a further five times. Fifty μL of TMB substrate (3,3/, 5, 5/-tetramethyl-benzidine
19
liquid substrate system) was added to develop the reaction. Fifty μL of 0.2 M sulfuric acid
20
was added to stop the reaction, and the plate was read in a ELISA plate reader at an optical
21
density 450/570 nm.
22
Monoclonal antibodies (omalizumab and QGE031, both at 5 μg/mL) in 10 mM
23
sodium acetate (pH 4.5) were immobilized at a flow rate of 5 μL/min on a CM5 biosensor
24
chip by standard amine coupling with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
3
1
hydrochloride, N-hydroxy-succinimide and ethanolamine hydrochloride. Different
2
concentrations of the IgE analytes from three species (human, cynomolgus monkey and
3
rhesus monkey) were injected onto the chip at a flow rate of 30 μL/min over 3–5 min and
4
allowed to dissociate for 30 min. The chip surface was regenerated at 100 μL/min with 100
5
μL 100 nM hydrochloric acid. A reference flow cell (phosphate buffered saline) was also
6
prepared. A blank (phosphate buffered saline) was injected under the same conditions as the
7
IgE analytes. Biacore software was used to determine the cross-species binding.
8
9
Methods
10
Clinical trials
11
Dose selection:
12
The intravenous study was the first-in-human study and was therefore conducted without
13
prior information of the pharmacokinetics or pharmacodynamics of QGE031 in humans. The
14
starting dose of 0.1 mg/kg was chosen based on several factors. Firstly, the 12-fold difference
15
in dissociation constant from non-human primates where doses up to 120 mg/kg were given
16
allowed for a possible starting dose of 10 mg/kg. A standard safety factor of 10-fold when
17
extrapolating from animals to man reduced the starting dose to 1 mg/kg. Finally, when
18
comparing the in vitro suppression of free IgE by QGE031 with that of an anti-IgE of similar
19
affinity, HAE-1 [2], the dose of QGE031 that would provide a similar suppression was
20
estimated to be 10-fold lower, allowing a starting dose of 0.1 mg/kg. As this was a first-in-
21
human study, the first two subjects in Cohort 1 were dosed on consecutive days in an open-
22
label manner. The remaining subjects in Cohort 1 were dosed in a double-blind manner. For
23
subsequent Cohorts (2–6), subjects were dosed in a double-blind manner with at least 2 h
24
separating the dosing of each subject.
4
1
The highest dose of QGE031 for the subcutaneous study was based upon model-based
2
pharmacokinetics simulations that predicted an adequate exposure margin compared with
3
exposure to non-human primates in preclinical safety studies
4
Institutional Review Boards:
5
There was one Institutional Review Board (IRB) for the intravenous study (Aspire IRB, CA,
6
USA) and three IRBs for the subcutaneous study (Aspire IRB, CA, USA; Quorum Review
7
Inc, WA, USA and Alpha IRB, CA, USA).
8
9
Protocol amendments:
10
Intravenous trial
11
The study protocol was amended five times, and most amendments included administrative
12
and personnel changes. None of the amendments were considered to have affected the
13
interpretation of study results as they occurred prior to unblinding. Of the five amendments,
14
the most significant was the introduction of the placebo expansion group (Cohort 6a);
15
Amendment 5, 13 November 2009.
16
17
Subcutaneous trial
18
The protocol was amended once after a planned interim analysis that revealed no urticarial
19
events in at least 36 subjects who had received four doses of QGE031 (Amendment 1, 02
20
February 2011). The amendment included an additional cohort of subjects who were to
21
receive a higher dose of QGE031 (Cohort 6) to further explore the safety, tolerability,
22
pharmacokinetics and pharmacodynamics of QGE031 to facilitate dose selection for
23
subsequent Phase II studies. The amendment also modified the visit schedule of subjects who
24
received 4-weekly doses of omalizumab. The original version of the protocol assumed
5
1
biweekly dosing of omalizumab. However, several subjects were dosed monthly in
2
accordance with the established dosing table [3] and the modified visit schedule streamlined
3
visits and assessments on non-dosing days. The amendment allowed inclusion of subjects
4
with atopic dermatitis to facilitate recruitment of subjects with IgE > 700 IU/mL.
5
6
Pharmacokinetic measurement of QGE031 in serum:
7
One hundred microliters of 2 μg/mL biotin conjugated mouse-anti-human-IgE antibody (BD
8
Pharmingen No. 555858, Batch 07164, 0.5 mg/mL) was coated onto each well of
9
streptavidin-precoated 96-well microplates (F96, No. 436014, Nunc). The plates were placed
10
on a microplate shaker (Titramax 1000, Heidolph, Merck) and incubated in the dark at room
11
temperature for 1 h. The plates were then washed three times with 300 μL/well of wash
12
buffer (Tris Buffered Saline with Tween-20® pH 8.0, No. T-9039, Sigma). Ten microliters of
13
standard samples, unknowns and quality controls were added to 475 μL of sample dilution
14
buffer (Low Cross Buffer, Pierce), together with 25 μL of 100 μg/mL of human-IgE solution
15
(Myeloma Biodesign, A50796H, batch 1E14405, 1.199 mg/mL). After shaking for 30 min at
16
room temperature, 100 μL was added to each well. The plates were placed on the microplate
17
shaker and incubated for 2 h in the dark at room temperature.
18
The plates were washed four times with wash buffer to remove any unbound
19
substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal
20
antibody specific for human IgG (1:10000 dilution of 1.8 mg/mL, No. A2064, Lot
21
077K4793, Clone CG-5, Sigma) was added to bind to the captured QGE031, and the plate
22
was again incubated (shaking) for 1 h in the dark at room temperature. After three additional
23
washing steps to remove unbound reagent, 100 μL of substrate solution (No. UP664791,
24
Uptima) was added and incubated for 50 min (shaking) in the dark at room temperature. The
6
1
plates were read using a Vmax Microplate reader (Molecular Devices) at 405 nm using
2
SoftMax Pro (version 5.0.1) software.
3
4
Pharmacodynamic assessments - total IgE:
5
One hundred microliters of 0.5 μg/mL biotin conjugated goat-anti-human-IgE antibody (Ref.
6
A80-108A, Bethyl) was coated onto each well of streptavidin-precoated 96-well microplates
7
(F96, No. 436014, Nunc) and placed on a microplate shaker (Titramax 1000, Heidolph,
8
Merck) and incubated in the dark at room temperature for 1 h. The plates were then washed
9
three times with 300 μL/well of wash buffer (Tris Buffered Saline with Tween-20® pH 8.0,
10
No. T-9039, Sigma). One hundred microliters of standard samples, unknowns and quality
11
controls were added to 100 μL of a solution of QGE031 (300 μg/mL) and shaken for 30 min
12
at room temperature to complex all the IgE in the sample. The complexes were then further
13
diluted 1:10 with Low Cross Buffer (Pierce) and 100 μL added to each well followed by
14
incubation (shaking) for 2 h at room temperature in order to capture the complex with the
15
coated goat-anti-IgE antibody.
16
The plates were washed three times with wash buffer to remove any unbound
17
substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal
18
antibody specific for human IgG (1:10000 dilution of 1.8 mg/mL, No. A2064, Lot
19
077K4793, Clone CG-5, Sigma) was added to bind to the captured QGE031 and the plate
20
was again incubated (shaking) for 1 h in the dark at room temperature. After three additional
21
washing steps to remove unbound reagent, 150 μL of substrate solution (No. UP664791,
22
Uptima) was added and incubated for 60 min (shaking) in the dark at 37°C. Optical density
23
was measured using a Vmax microplate reader (Molecular Devices) at 405 nm and using
24
SoftMax Pro (version 5.0.1) software.
7
1
2
Pharmacodynamic assessments - free IgE:
3
One hundred microliters of IgE high-affinity receptor (FcεRI conjugated, 1.5 μg/mL,
4
Novartis Pharma AG) was added to each well of 96-wells microplates (MaxiSorp plates, No.
5
439454, Nunc) and incubated over night at 4°C. The plates were then washed four times with
6
300 μL/well of wash buffer (Tris Buffered Saline with Tween-20® pH 8.0, No. T-9039,
7
Sigma). Three hundred microliters of Superblock Blocking Buffer (No. 37535, Pierce) was
8
added to each well and the plate was placed on a microplate shaker (Titramax 1000,
9
Heidolph, Merck) and incubated for 20 min at room temperature. The plates were then
10
washed a further four times with wash buffer. Each standard sample, unknowns and quality
11
controls were diluted 1:5 (Low Cross Buffer, Pierce) and 100 μL of diluted sample was added
12
to each well and incubated for 2 h (shaking) at room temperature in the dark.
13
The plates were washed four times with wash buffer to remove any unbound
14
substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal
15
antibody specific for human IgE (No. 555859, BD) was added and the plate was again
16
incubated (shaking) for 1 h in the dark at room temperature. After six washing steps to
17
remove unbound reagent, 50 μL of substrate solution NADPH was added to each well and
18
incubated for 15 min (shaking) at room temperature. Finally, 50 μL of Amplifier solution
19
(No. 19589-019, Invitrogen) was added to each well and then incubated (shaking) for 20 min
20
at room temperature. Fifty microliters of Stop solution (Sulfuric acid) was added to terminate
21
the reaction. Optical density was measured using a VersaMax microplate reader (Molecular
22
Devices) at 495 nm using SoftMax Pro (Version 5.0.1) software.
23
24
8
1
Pharmacodynamic assessments: fluorescence-activated cell sorting analysis:
2
Antibodies and reagents
3
Basophil selection: Mouse anti-human Lineage cocktail-FITC, monoclonal antibody cocktail-
4
FITC (BD 340546), Mouse anti-Human CD203c-PerCp-Cy5.5 monoclonal antibody
5
(BioLegend 324608). For the expression levels of FcεRI and IgE: Mouse monoclonal anti-
6
Human FcεRI-APC (eBioscience 17-5699), mouse monoclonal anti-human IgE-PE
7
(BioLegend 325506).
8
9
Protocol
10
At the clinical site antibody mixes were added to 200 µL aliquots of whole blood and
11
incubated for 30 min at room temperature. Lyse/fix buffer (BD 349202) was then added and
12
the samples frozen at -80°C and sent to the fluorescence-activated cell sorting analysis
13
laboratory (Cellular Biomarker Group, Novartis Institutes for Biomedical Research, Basel,
14
Switzerland). After thawing and antibody-staining with the antibodies mentioned above,
15
2000 basophils were acquired for each sample by flow cytometry performed on a
16
fluorescence-activated cell sorting Canto II cytometer. Molecules of equivalent soluble
17
fluorochrome values were calculated using Quantum molecules of equivalent soluble
18
fluorochrome beads (Bangs Laboratories Inc.)
19
20
Pharmacokinetic and pharmacodynamic model of binding to and capture of IgE by QGE031
21
and omalizumab:
22
The pharmacokinetic profile of QGE031 and several pharmacodynamic parameters including
23
total IgE, basophil FcRI and basophil surface IgE were analyzed using an adaptation of the
24
previously published omalizumab PK-IgE binding model [4-6]. The published model was
9
1
updated to include: i) two compartments instead of one to allow the analysis of the
2
intravenous pharmacokinetics of QGE031; ii) basophil FcεRI and surface IgE as indirect
3
responses to free IgE suppression; iii) both QGE031 and omalizumab with the assumption
4
that the only difference between the compounds was their ability to bind IgE given that they
5
are both IgG1 monoclonal antibodies. Model parameters for drug clearances and volumes of
6
distribution, binding of QGE031 or omalizumab to and turnover of IgE, the effect of free IgE
7
on basophil FcRI and surface IgE were estimated to get the mathematical equations to fit the
8
clinical time course data for the pharmacokinetic and pharmacodynamic assessments. As in
9
prior publications, the NONMEM® software (Icon plc, Dublin, Ireland) was used for the
10
model based analysis.
11
12
Pharmacodynamic assessments: extinction skin prick testing (subcutaneous trial):
13
At screening, skin prick testing was performed with single doses of multiple allergens.
14
Subsequent extinction skin prick testing was performed in duplicate on the subjects’ skin of
15
the back with serial three-fold dilutions of a selected allergen that provided a > 5 mm mean
16
wheal diameter at screening. Extinction skin prick testing was performed at baseline, on Days
17
29, 57 and 85, and at the end of the study (Day 155). Wheal and flare reactions were outlined
18
by pen and transferred by tape to a permanent record; the mean of the longest diameter and
19
corresponding midpoint orthogonal diameter were recorded. Only the flares were measured
20
for subjects in Cohort 1, which was a protocol deviation. Data from one site were excluded
21
due to poor duplicates and lack of a dose-response curve. Data from two subjects at Day 85
22
were excluded from the analyses due to protocol deviations (administration of antihistamines
23
prior to skin prick testing). These decisions were taken prior to database lock and unblinding
24
of the trial.
10
1
2
Statistical methods - skin prick data:
3
Arithmetic means of the skin prick test wheal and flare diameters were calculated from the
4
longest measurement and its orthogonal diameter, performed in duplicate. A positive
5
response for each dilution was defined where the mean diameter was at least 3 mm greater
6
than the mean diameter for the negative control. The threshold (i.e., the maximum dilution
7
level eliciting a positive wheal and flare response) was derived for each subject. Values were
8
assigned according to the dilution level (i.e., a value of 1 for the first dilution, 1:3, a value of
9
2 for the second dilution, and so on to a highest value of 10 for a dilution of 1:59,049). If a
10
response was seen with neat allergen the maximum dilution was assigned a value of 0. If no
11
positive skin prick test occurred the maximum dilution level was assigned a value of -1.
12
.
11
1
References
2
1 Nechansky A, Robertson MW, Albrecht BA, Apgar JR, Kricek F. Inhibition of antigen-
3
induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI
4
alpha-chain receptor antibody: implications for the involvement of the membrane-
5
proximal alpha-chain region in Fc epsilon RI-mediated cell activation. J Immunol 2001;
6
166:5979–90.
7
2 Putnam WS, Li J, Haggstrom J et al. Use of quantitative pharmacology in the
8
development of HAE1, a high-affinity anti-IgE monoclonal antibody. AAPS J 2008;
9
10:425–30.
10
11
12
3 Genentech. Xolair: US prescribing information. Updated July 2010. Last accessed 15
February 2014. http://www.gene.com/download/pdf/xolair_prescribing.pdf
4 Hayashi N, Tsukamoto Y, Sallas WM, Lowe PJ. A mechanism-based binding model for
13
the population pharmacokinetics and pharmacodynamics of omalizumab. Br J Clin
14
Pharmacol 2007; 63:548–61.
15
5 Lowe PJ, Tannenbaum S, Gautier A, Jimenez P. Relationship between omalizumab
16
pharmacokinetics, IgE pharmacodynamics and symptoms in patients with severe
17
persistent allergic (IgE-mediated) asthma. Br J Clin Pharmacol 2009; 68:61–76.
18
6 Slavin RG, Ferioli C, Tannenbaum SJ, Martin C, Blogg M, Lowe PJ. Asthma symptom
19
re-emergence after omalizumab withdrawal correlates well with increasing IgE and
20
decreasing pharmacokinetic concentrations. J Allergy Clin Immunol 2009; 123:107–13.
21
22
12
Table S1. Subject demographics for treatment groups for (a) the intravenous trial and (b) the subcutaneous trial
(a)
Cohort 1
Cohort 2
Cohort 3
Cohort 4
Cohort 5
Cohort 6
Cohort 7
0.1 mg/kg
0.3 mg/kg
1.0 mg/kg
3.0 mg/kg
3.0 mg/kg
10 mg/kg
Xolair
Placebo
Cohort 6a
Placebo
All
treatments
(expansion
IgE
group)
> 1000
IU/mL
n=6
n=6
n=8
n=9
n=1
n=6
n=8
n = 11
n = 18
N = 73
Mean (SD)
32.3 (11.7)
31.5 (9.4)
39.3 (10.5)
35.0 (8.3)
29.0
30.7 (9.4)
34.3 (8.6)
34.5 (12.5)
32.9 (8.7)
33.8 (9.6)
Range
20, 48
22, 47
25, 50
19, 46
NC
22, 48
24, 50
18, 48
22, 51
18, 51
6 (100)
6 (100)
5 (63)
9 (100)
1 (100)
6 (100)
8 (100)
8 (73)
17 (94)
66 (90)
76.1 (3.1)
69.7 (12.5)
81.2 (15.1)
81.4 (13.0)
77.9
80.8 (7.5)
81.4 (10.2)
75.6 (14.2)
78.6 (10.8)
78.3 (11.5)
Age (years)
Gender
Male, n (%)
Weight (kg)
Mean (SD)
13
Range
72.9, 80.1
60.0, 87.5
Mean (SD)
173.2 (6.9)
Range
56.6, 99.4
59.7, 99.8
NC
71.3, 92.0
68.6, 100.0
58.0, 99.2
50.4, 93.5
50.4, 100.0
172.7 (10.8) 170.6 (12.8)
176.1 (5.4)
182
175.8 (6.2)
175.5 (4.7)
173.4 (6.8)
173.1 (7.6)
173.8 (7.7)
166, 185
159, 189
154, 193
169, 187
NC
166, 184
170, 182
159, 181
160, 187
154, 193
Caucasian
4 (67)
3 (50)
5 (63)
6 (67)
0
5 (83)
6 (75)
6 (55)
11 (61)
46 (63)
Black
2 (33)
3 (50)
3 (38)
3 (33)
1 (100)
0
2 (25)
3 (27)
1 (6)
18 (25)
Asian
0
0
0
0
0
1 (16.7)
0
1 (9.1)
5 (27.8)
7 (9.6)
Other
0
0
0
0
0
0
0
1 (9.1)
1 (5.6)
2 (2.7)
Height (cm)
Race, n (%)
SD, standard deviation.
14
(b)
Cohort 1
Cohort 2
Cohort 3
Cohort 4
Cohort 5
Cohort 6
and 6 pooled
IgE > 700 IU/mL
0.2 mg/kg
Placebo
0.6mg/kg
2 mg/kg
2 mg/kg
Placebo
Cohorts 2, 3
Omalizuma
4 mg/kg
placebo*
b
n=8
n=4
n=8
n = 40
n=6
n=6
n = 12
n=8
n = 18
Mean (SD)
39 (10.2)
41 (12.0)
28 (9.7)
33 (9.4)
33 (7.5)
32 (11.8)
33 (10.3)
37 (11.5)
35 (11.4)
Range
23, 49
24, 52
20, 49
19, 53
26, 43
19, 48
20, 55
25, 53
19, 55
4 (50)
4 (100)
7 (88)
35 (88)
5 (83)
5 (83)
10 (83)
7 (88)
15 (83)
Mean (SD)
81.4 (20.3)
69.2 (7.0)
76.7 (15.8)
82.1 (13.3)
91.5 (18.9)
83.3 (22.5)
86.1 (11.5)
81.8 (14.8)
80.0 (15.0)
Range
61.0, 112.6
61.0, 76.5
61.7, 100.7
63.3, 114.6
68.9, 116.5
59.1, 118.4
70.2, 111.0
64.0, 106.4
49.1, 103.5
Age (years)
Gender
Male, n (%)
Weight (kg)
Height (cm)
15
Mean (SD)
171 (9.5)
170 (7.0)
179 (9.6)
176 (7.3)
177 (4.3)
170 (4.5)
175 (7.8)
174 (8.7)
173 (11.6)
Range
156, 188
161, 177
166, 195
163, 195
169, 181
165, 178
163, 187
160, 186
151, 192
Caucasian
5 (63)
3 (75)
5 (63)
25 (63)
3 (50)
4 (67)
8 (67)
6 (75)
12 (67)
Black
2 (25)
1 (25)
3 (38)
12 (30)
3 (50)
1 (17)
4 (33)
1 (13)
4 (22)
Asian
0
0
0
1 (3)
0
1 (17)
0
1 (13)
1 (6)
Other
1 (13)
0
0
2 (5)
0
0
0
0
1 (6)
Race, n (%)
*Pooled over Cohorts 2, 3 and 6.
SD, standard deviation.
16
Table S2. Statistical analysis of the wheal area under the curve from the skin prick testing in
the subcutaneous study
N*
LS mean
Difference
95% CI
P-value
compared
with placebo
Day 29
Cohort 2 (0.6 mg/kg)
4
19.3
-9.8
-20.1, 0.5
0.063
Cohort 3 (2 mg/kg)
29
15.1
-14.0
-19.7, -8.3
< 0.001
Cohort 6 (4 mg/kg)
7
18.4
-10.7
-19.1, -2.3
0.013
Cohorts 2, 3 and 6 (pooled placebo)
16
29.1
Cohort 4 (2 mg/kg IgE > 700 IU/mL)
5
13.5
-23.6
-35.8, -11.4
< 0.001
Cohort 4 (placebo IgE > 700 IU/mL)
4
37.1
Cohort 2 (0.6 mg/kg)
4
13.2
-21.1
-33.6, -8.5
0.001
Cohort 3 (2 mg/kg)
27
8.0
-26.3
-33.3, -19.3
< 0.001
Cohort 6 (4 mg/kg)
7
11.8
-22.5
-32.7, -12.3
< 0.001
Cohorts 2, 3 and 6 (pooled placebo)
16
34.3
Cohort 4 (2 mg/kg IgE > 700 IU/mL)
4
-0.1
-38.8
-55.9, -21.8
< 0.001
Cohort 4 (placebo IgE > 700 IU/mL)
3
38.8
Cohort 2 (0.6 mg/kg)
4
14.9
-19.6
-34.6, -4.5
0.012
Cohort 3 (2 mg/kg)
26
0.4
-34.1
-42.7, -25.4
< 0.001
Cohort 6 (4 mg/kg)
7
4.7
-29.7
-41.9, -17.6
< 0.001
Cohorts 2, 3 and 6 (pooled placebo)
14
34.4
Cohort 4 (2 mg/kg IgE > 700 IU/mL)
5
9.1
-31.8
-50.8, -12.9
0.001
Day 57
Day 85
17
Cohort 4 (placebo IgE > 700 IU/mL)
3
40.9
Cohort 2 (0.6 mg/kg)
4
52.1
10.7
-17.5, 38.9
0.45
Cohort 3 (2 mg/kg)
31
17.5
-23.8
-39.3, -8.4
0.003
Cohort 6 (4 mg/kg)
8
22.1
-19.2
-41.3, 2.8
0.086
Cohorts 2, 3 and 6 (pooled placebo)
16
41.3
Cohort 4 (2 mg/kg IgE > 700 IU/mL)
5
32.1
-7.7
-41.3, 26.0
0.65
Cohort 4 (placebo IgE > 700 IU/mL)
4
39.8
Day 155 (end of study)
*Analysis included only those subjects who had a value at both baseline and post-dose.
LS, least-squares; CI, confidence interval.
18
Figure legends
Fig. S1. Overview of the pharmacokinetics of QGE031 and pharmacodynamic responses to
QGE031, omalizumab or placebo for total and free IgE, basophil FcεRI and surface IgE, skin
prick test wheal and flare responses.
Red lines indicate patients with IgE screening levels > 1000 IU/mL (3 mg/kg) or
> 700 IU/mL (placebo, 2 mg/kg QGE031). For comparative puprposes across studies,
placebo and omalizumab groups contain subjects pooled from both trials.
Data presented are individual subject responses.
iv, intravenous; MESF, molecules of equivalent soluble fluorochrome; s, soluble; sc,
subcutaneous.
Fig. S2. Percentage of subjects treated with QGE031 (0.6, 2, 4 mg/kg), omalizumab and
placebo with positive wheal responses by dilution level following skin-prick challenge.
19