Methods
Hydrogen/Deuterium Exchange Mass Spectrometry
For the protein and antibody sample, CD33 and antibody (BI 836858 or HuM195) were
incubated for 15 minutes at room temperature. The final molar ratio antibody to CD33 was 2:1.
Using a LEAP robot system (exchange plate kept at 250C, sample/quench plate kept at 4oC), 8
µl of sample was added to 80µl of exchange buffer (10mM NaH 2 PO 4 in D2O, pH=7 or 10mM
NaH2PO4 in H2O, pH=7), mixed, and allowed to exchange for various times (15, 60, 120, 240,
and 600 seconds) at 250C. 80µl of this solution was then transferred to 80µl of quench buffer
(1M urea, 0.1M TCEP-HCl) at 4oC and mixed. 90µl of this solution was then transferred to 10µl
of pepsin (4 mg/ml) at 40C and mixed. After 2 minutes, 60µl of this solution was injected onto a
C18 trap cartridge. The cartridge was washed with H 2 O + 0.1%TFA for 2 minutes at 100µl/min.
A valve was then switched and the cartridge eluted onto a Phenomenex Jupiter C5 column, 1.0
x 50mm, 5µm, 300A. Mobile Phase A was water/acetonitrile/TFA (99/1/0.05) and Mobile Phase
B was acetonitrile/water/TFA (95/5/0.05). Flow rate was 100µl/min. Gradient was: 0 minutes
(0%B), 6 minutes (40%B), 7 minutes (40%B), 8 minutes (90%B), 10 minutes (90% B), 11
minutes (0%B). The LEAP system precools the mobile phase to 4oC and also maintains the
trap column and analytical column at 4oC.
For the mass spectroscopy (MS) experiments a single scan method from 300-2000 for 14
minutes was used at resolution 60,000. For the MS/MS experiments a method with 7 scans was
used for 14 minutes. The first scan was a full range scan from 300-2000 at 30,000 resolution.
Subsequent scans were CID scans of the 6 most intense ions from scan #1. Isolation width was
1.5amu, collision energy was 35V, and activation time was 30msec. Pepsin peptides were
identified using fragmentation data and the program Proteome Discoverer (Thermo). Identified
peptides were analyzed using an in-house program which calculates the average mass for
exchanged peptides.
Internalization assay
HL60 cells (5x105 cells/ml) were incubated with either BI 836858 or HuM195 for 24h, 4h or 1h
with a final antibody concentration of 10µg/ml. To determine the baseline value for CD33
expression, some wells were incubated with 50µl cell culture medium without antibody. After
incubation cells were washed twice with 200µl PBS/FCS 3% and 0.09% sodium azide to stop
internalization. Thereafter cells were stained with mAb (10µg/ml) followed by PE-conjugated
secondary antibody (anti-human IgG R-PE conjugate). Cells were washed to remove unbound
mAb and analyzed on a BD Biosciences FACS Canto Flow Cytometer. MFI values of untreated
cells were defined as reference (100% surface CD33). MFI values obtained from treated cells
were displayed as % residual CD33 expression.
Supplementary material, Figure 1: Panel A shows higher surface expression of CD33 when
with pre-treatment of HL60 cells with non-Fc engineered anti-CD33 antibody (BI836854) in
comparison to Lintuzumab. Panel B shows that Fc-engineering increases ADCC. Human
leukemia HL60 cells were incubated with BI 836854 (non Fc-engineered parental mAb of BI
836858) and BI 836858, and ADCC was determined as described in ref. 19. Effector cells
(PBMC) were derived from a healthy donor, the effector to target cell ratio was 25:1.
Supplementary Figure 2: Optimization of BI 836858 concentration and effector to target
(E: T) ratio for ADCC induction. Human leukemia HL60 cells were pre-coated with different
concentrations of BI 836858, and then co-cultured with NK cells enriched from two healthy
donors at several effector to target (E:T of 3:1, 6.25:1, 12.5:1 and 25:1) ratios for the ADCC
assay. Negative control antibody (Herceptin) at the highest E: T ratio (25:1) was included as
negative control (labeled as “25her”).
Supplementary table 1: Binding of BI 836858 to AML cell lines and primary AML blasts
Supplementary table 2: Characteristics of AML patients whose blasts were used in the
ADCC and CD107a assays.
Supplementary table 3: Fold expression of NKG2DL as measured by RT-PCR in bone
marrow aspirates in 17 patients that received a 10-day infusion of decitabine. Data were
normalized to internal control GAPDH. Normalized data (dct) were analyzed by using mixed
effect model, incorporating repeated measures for each subject. The primary test results
(difference in gene expression between post-28 and pre-decitabine treatment) are summarized.
Supplementary material, Figure 1: Panel A shows higher surface expression of CD33 when
with pre-treatment of HL60 cells with non-Fc engineered anti-CD33 antibody (BI836854) in
comparison to Lintuzumab. Panel B shows that Fc-engineering increases ADCC. Human
leukemia HL60 cells were incubated with BI 836854 (non Fc-engineered parental mAb of BI
836858) and BI 836858, and ADCC was determined as described in ref. 19. Effector cells
(PBMC) were derived from a healthy donor, the effector to target cell ratio was 25:1.
A
100
B
80
BI 836858
BI 836854
% C y to ly s is
60
40
20
0
-2 0
-4 0
-2
-1
0
1
lo g [m A b ] ( n g /m l)
2
3
4
Supplementary material, Figure 2. Optimization of BI 836858 concentration and effector
to target (E: T) ratio for ADCC induction.
Supplementary material, Table 1: Binding of BI 836858 to AML cell lines and primary AML
blasts
Cell line
K D (nM)
Binding Sites/cell
HL-60
1.0E-09
15602
Molm-13
5.2E-09
50351
GF-D8
3.1E-09
75628
KG-1
1.9E-09
20540
HEL92.1.7
2.9E-10
56871
TF-1
1.1E-09
77874
Sample #
K D (M)
Binding Sites/cell
1 (PB)
1.4E-09
5164
2 (PB)
4.2E-09
1423
3 (PB)
4.6E-09
6623
4 (BM)
2.3E-09
4855
5 (BM)
1.0E-10
5329
6 (PB)
1.7E-10
4905
7 (BM)
1.6E-09
6479
8 (PB)
1.8E-09
6035
9 (PB)
1.4E-09
7703
Supplementary material, Table 2. Characteristics of AML patients whose blasts were
used in the ADCC and CD107a assays.
Patient
% Bone WBC
Number
marrow
at DeNovo
diagnosis
FAB
FLT3 status
Classification
blast
AML 1
26%
102.6 K/µL
Yes
AML with MDS
Not tested
changes
AML 2
52%
228.0 K/µL
Yes
M2
ITD positive
AML 3
88%
147.7 K/µL
Yes
M1
ITD positive
AML 4
N/A
62.1 K/µL
Yes
M2
Not tested
AML 5
91%
198.0 K/µL
Yes
M1
Negative
AML 6
98%
213.7 K/µL
Yes
M2
Negative
AML 7
76%
232.5 K/µL
Yes
M5b
Negative
AML 8
92%
249.1 K/µL
No
M1
ITD positive
AML 9
50%
174.7 K/µL
Yes
AML with
Negative
inversion 16
AML 10
79%
287.8 K/µL
Yes
M1
ITD positive
AML 11
90%
139.8 K/µL
Yes
M1
ITD positive
AML 12
86%
176.4 K/µL
Yes
M5a
ITD positive
Supplementary material, Table 3. Fold expression of NKG2DL as measured by RT-PCR in
17 patients that received a 10-day infusion of decitabine.
Data were normalized to internal control GAPDH. Normalized data (dct) were analyzed by
using mixed effect model, incorporating repeated measures for each subject. The primary test
results (difference in gene expression between post-28 and pre-decitabine treatment) are
summarized.
Gene
Fold (Day28 vs Day1)
p-value
95% CI*
ULBP1
2.17
0.0069
(1.25, 3.73)
ULBP2
2.88
0.0016
(1.54, 5.37)
ULBP3
2.04
0.0101
(1.2, 3.45)
MICA
1.53
0.0547
(0.99, 2.4)
MICB
1.70
0.0323
(1.04, 2.75)
PVR
0.82
0.5804
(0.39, 1,71)
NECTIN2
1.13
0.5286
(0.77, 1.66)