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SUPPLEMENTAL DATA
Supplementary Information for Cardillo et al. (Synthetic lethality exploitation by an anti-Trop-2-
SN-38 antibody-drug conjugate, IMMU-132, plus PARP-inhibitors in BRCA1/2-wild-type triple-
negative breast cancer)
Table of Contents
Supplementary Data PgIn vitro combination cytotoxicity assays 2Western blot assessment of PARP activity, double-stranded DNA breaks, and HRR protein expression in vitro 3
Cell cycle analysis 4Hematologic toxicity of IMMU-132 plus olaparib in naïve BALB/c mice 4Table S1: Summary of the percentage of TNBC cells in various phases of cell cycle when
treated with the combination of rucaparib and IMMU-132. 6
Table S2: Summary of the percentage of TNBC cells in various phases of cell cycle when treated with the combination of talazoparib and IMMU-132. 7
Table S3: Area under the curve comparisons between IMMU-132 (250 g) plus olaparib (1 mg) treated HCC1806 tumor-bearing mice and all other treatment groups. 8
Table S4: Area under the curve comparisons between IMMU-132 plus olaparib treated MDA-MB-468 tumor-bearing mice and all other treatment groups. 9
Figure Legends 10Fig. S1. Western blot of HRR proteins in TNBC cell lines exposed to IMMU-132. 12Fig. S2. Isobolograms of normalized IC50-values of IMMU-132 plus PARPi in various TNBC
tumor-lines. 13
Fig. S3. Cell cycle analysis of various TNBC cell lines incubated with IMMU-132 and olaparib. 14
Fig. S4. Tolerability of IMMU-132 Combined with olaparib or talazoparib in athymic nu/nu nude mice. 15
References 16
1
In vitro combination cytotoxicity assays
The in vitro cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium dye reduction assay (MTS dye
reduction assay; Promega, Madison, WI). Briefly, cells were plated into 96-well clear, flat-
bottomed plates overnight. Dose-response curves for each agent alone were first tested to
determine single agent IC10, IC20, or IC30–values after 96-h incubation. In combination assays,
one agent (e.g., IMMU-132) is tested on a given cell line across a range of concentrations (i.e.,
dose-response curves) either alone or in the presence of a constant amount (IC 10, IC20, or IC30–
concentration) of the second agent (e.g., olaparib). This is then reversed in which the PARPi
was plated across a concentration range either alone or with a constant amount of IMMU-132
(i.e., IC10, IC20, or IC30–concentration). Growth inhibition was measured as a percent of growth
relative to untreated cells. Dose-response curves were generated from the mean of triplicate
determinations, and IC50-values were calculated using Prism GraphPad Software (v6.05)
software package (Advanced Graphics Software, Inc.; Encinitas, CA). For each condition, the
IC50-value was determined from these data. Combinatorial Index (CI) numbers were calculated
using the following formula: CI=Da/Dxa + Db/Dxb (1),
where,
Da=IC50 of IMMU-132 when used in combination with a constant amount of PARPi (IC10, IC20, or
IC30 concentration);
Db=IC50 of PARPi when used in combination with a constant amount of IMMU-132 (IC10, IC20, or
IC30 concentration);
2
Dxa=IC50 of IMMU-132 when used alone; and
Dxb=IC50 of PARPi when used alone.
(Antagonistic Effect CI >1.0,
Additive Effect CI = 1.0,
Synergistic Effect CI<1.0).
Western blot assessment of PARP activity and double-stranded DNA breaks in vitro
Cells were lysed in Ripa buffer containing protease/phosphatase inhibitor cocktail (Cell Signaling
Technology) and protein concentrations were determined using BCA Protein Assay Kit
(ThermoFisher). For PAR and PARP detection, 60 µg of protein was resolved using 3-8% Tris-
Acetate NuPAGE gel and transferred to PVDF using Tris-Glycine transfer buffer containing 10%
methanol and 0.02% SDS. For phosphorylated-H2A.X (p-H2A.X) and β-actin detection, 60 µg of
protein was resolved using 4-12% Bis-Tris NuPAGE gels and transferred to nitrocellulose using
Tris-Glycine transfer buffer containing 20% methanol. Primary and secondary antibodies were
purchased from Cell Signaling Technology unless otherwise noted and include anti-phospho
H2A.X (Cat. No. 2577), anti- PAR (EMD Millipore, Cat. No. MABC547), anti-PARP (Cat. No. 9532),
and anti-β-actin (Cat. No. 4967). Secondary antibodies were horseradish peroxidase conjugated
antibodies to mouse (Cat. No. 7076) or rabbit (Cat. No. 7074) IgG. SuperSignal ®West Dura
Extended Duration Substrate (Thermo Scientific) was used for detection. For HRR proteins,
protein lysate (30µg) was loaded on a 4-12% Bis-Tris NuPAGE gel and transferred to PVDF using
NuPAGE transfer buffer containing 10% methanol. Primary and secondary antibodies were
3
purchased from Cell Signaling Technology unless otherwise noted and include anti-ERCC1 (Cat.
No. 12345), anti-Rad51 (Cat. No. 8875), anti-BARD1 (abcam, Cat. No. ab50984), anti-FANCD2 (abcam,
Cat. No. ab108928) and anti-β-actin (Cat. No. 4967). Secondary antibodies were horseradish
peroxidase-conjugated antibodies to rabbit (Jackson Immunoresearch, Cat. No. 111-035-046) or
mouse (Jackson Immunoresearch, Cat. No. 115-035-003). SignalFire™ ECL Reagent (Cell
Signaling Technology) was used for detection.
Cell cycle analysis
Asynchronistic cells were harvested from tissue culture and plated in 6-well plates overnight
(1X106 cells/well). Test agents (IMMU-132 and PARPi) were added to appropriate wells at
concentrations indicated in the figure legends. Plates were then placed back in the incubator
for 24 h after which cells were harvested and cell-cycle analysis performed via FACS using BD
Cycletest™ Plus DNA Kit (Becton-Dickinson, Cat. 340242) on a BD FACSCanto FACS machine.
Hematologic toxicity of IMMU-132 plus olaparib in naïve BALB/c mice
Female BALB/c mice (10 weeks old) were divided into four different treatment groups of 10
mice each. Starting on day 1, groups of mice received olaparib (50 mg/kg i.p., days 1-5 and 8-
12), IMMU-132 (250 g i.v., days 1 and 8), or the combination of IMMU-132 plus olaparib.
Control animals receive saline (100 L i.v. days 1 and 8) and 2-hydroxy-propyl--cyclodextran/PBS
(10% w.v., 200 L i.p. days 1-5 and 8-12). Five mice from each group were anesthetized and
bled via intracardiac puncture using heparinized syringes on day 15. On day 22, the remaining 5
mice were likewise bled. Whole blood (200 L) was transferred to EDTA (purple cap) tubes and
4
sent to VRL Laboratories (Gaithersburg, MD) for automated complete blood count
determinations.
5
Table S1: Summary of the percentage of TNBC cells in various phases of cell cycle when treated with the combination of rucaparib and IMMU-132.
Cell LinesCell Cycle
Phase
Treatment
UntreatedIMMU-132
(5 nM*)Rucaparib
(1 M)
IMMU-132+
RucaparibHCC1806 G1 89.4 70.7 84.6 32.9
S 8.0 27.8 10.1 64.1G2/M 2.6 1.5 5.3 3.1
HCC38 G1 76.2 46.6 72.3 35.2S 15.9 38.4 17.0 42.2
G2/M 8.0 15.0 10.7 22.6MDA-MB-231 G1 79.8 81.4 79.4 66.0
S 13.2 14.8 13.3 28.4G2/M 7.0 3.8 7.3 5.6
MDA-MB-468 G1 83.3 55.5 84.1 50.4S 11.8 37.6 11.1 41.9
G2/M 4.9 6.9 4.8 7.8*SN-38 equivalents.
Table S2: Summary of the percentage of TNBC cells in various phases
6
of cell cycle when treated with the combination of talazoparib and IMMU-132.
Cell LinesCell Cycle
Phase
Treatment
UntreatedIMMU-132
(5 nM*)
Talazoparib
(0.2 M)
IMMU-132+
TalazoparibHCC1806 G1 89.4 70.7 87.4 35.3
S 8.0 27.8 10.2 61.9G2/M 2.6 1.5 2.5 2.8
HCC38 G1 76.2 46.6 49.3 33.2S 15.9 38.4 29.4 47.7
G2/M 8.0 15.0 21.2 19.1MDA-MB-231
G1 79.8 81.4 80.2 69.4S 13.2 14.8 14.7 26.6
G2/M 7.0 3.8 5.0 4.1MDA-MB-468
G1 83.3 55.5 82.7 50.1S 11.8 37.6 11.9 41.7
G2/M 4.9 6.9 5.3 8.2*SN-38 equivalents.
Table S3. Area under the curve comparisons between IMMU-132 (250 g) plus Olaparib (1 mg) treated HCC1806 tumor-bearing mice and all other treatment groups.
7
TreatmentsTime of
ComparisonTumor Volumes (cm3)
On that day (mean ± s.d.)P-Value(AUC)
IMMU-132 (250 g)plus
Olaparib
versus
IMMU-132 (250 g) Up to therapy Day 31 0.075 ± 0.105 vs. 0.599 ± 0.271 0.0002
Control ADC (250 g) + Olaparib (1 mg)
Up to therapy Day 24 0.051 ± 0.068 vs. 0.739 ± 0.221 <0.0001
Control ADC (250 g) Up to therapy Day 17 0.118 ± 0.067 vs. 0.840 ± 0.344 0.0001
Olaparib (1 mg) Up to therapy Day 14 0.162 ± 0.040 vs. 0.561 ± 0.326 0.0019
Saline Up to therapy Day 14 0.162 ± 0.040 vs. 0.646 ± 0.239 <0.0001
TreatmentsTime of
ComparisonTumor Volumes (cm3)
On that day (mean ± s.d.)P-Value(AUC)
IMMU-132 (250 g)
versus
Control ADC (250 g) + Olaparib (1 mg)
Up to therapy Day 24 0.420 ± 0.166 vs. 0.739 ± 0.221 0.0209
Control ADC (250 g) Up to therapy Day 17 0.331 ± 0.145 vs. 0.840 ± 0.344 0.0006
Olaparib (1 mg) Up to therapy Day 14 0.267 ± 0.092 vs. 0.561 ± 0.326 0.0051
Saline Up to therapy Day 14 0.267 ± 0.092 vs. 0.646 ± 0.239 <0.0001
8
Table S4. Area under the curve comparisons between IMMU-132 plus Olaparib treated MDA-MB-468 tumor-bearing mice and all other treatment groups.
TreatmentsTime of
ComparisonTumor Volumes (cm3)
on that day (mean ± s.d.)P-Value(AUC)
IMMU-132plus
Olaparib
versus
IMMU-132 Alone Up to therapy Day 56 0.186 ± 0.086 vs. 0.485 ± 0.277 0.0040
Control ADC plus Olaparib
Up to therapy Day 28 0.162 ± 0.044 vs. 0.545 ± 0.216 0.0010
Control ADC Alone
Up to therapy Day 35 0.120 ± 0.070 vs. 0.625 ± 0.259 0.0003
Olaparib Alone Up to therapy Day 28 0.162 ± 0.044 vs. 0.518 ± 0.250 0.0014
Saline Up to therapy Day 28 0.162 ± 0.044 vs. 0.690 ± 0.257 0.0001
TreatmentsTime of
ComparisonTumor Volumes (cm3)
on that day (mean ± s.d.)P-Value(AUC)
IMMU-132
versus
Control ADC plus Olaparib
Up to therapy Day 28 0.259 ± 0.074 vs. 0.545 ± 0.216 0.0098
Control ADC Alone
Up to therapy Day 35 0.265 ± 0.091 vs. 0.625 ± 0.259 0.0050
Olaparib Alone Up to therapy Day 28 0.259 ± 0.074 vs. 0.518 ± 0.250 0.0067
Saline Up to therapy Day 28 0.259 ± 0.074 vs. 0.690 ± 0.257 0.0005
9
Supplementary FIGURE LEGENDS
Figure S1. Western blot assessment of effects of IMMU-132 on expression of various
homologous recombination repair proteins (FANCD2, BARD1, Rad51, and ERCC1) in BRCA1/2
wild-type TNBC tumor-lines. Cells were plated overnight in 6-well plates before the addition
of IMMU-132 at the indicated concentrations (shown in SN-38 equivalents). After a 24-h
incubation, cells were harvested and cell lysates resolved and transferred for Western analysis
as described in Methods. BRCA1/2 wild-type cell lines are represented by MDA-MB-231, MDA-
MB-468, and BT-20. The HCC1806 BRCA1/2-defective TNBC cell line was used for comparison.
Figure S2. Isobolograms of normalized IC50-values of IMMU-132 plus PARPi in various TNBC
tumor-lines. Dose/response curves for each agent alone and in combination were generated
as described in Methods. These IC50-vlaues are then normalized and plotted as isobolograms.
Red dots indicate changes in IMMU-132 IC50 when in constant amount of a given PARPi (IC10,
IC20, and IC30). Blue dots indicate changes in a given PARPi IC50 when in a constant amount of
IMMU-132 (IC10, IC20, and IC30). Each condition was done in three independent assays.
Figure S3. Cell cycle analysis of various TNBC cell lines incubated with IMMU-132 and
olaparib. Asynchronistic cells were harvested from tissue culture and plated in a 6-well plate
overnight. Test agents (olaparib and IMMU-132) were added to appropriate wells and plates
incubated for 24 h. IMMU-132 concentrations are shown as SN-38 drug-equivalents. After 24 h,
cells were harvested and cell-cycle analysis performed via FACS as described in Methods.
Shaded areas indicate cells in S-phase of the cell cycle.
Figure S4. Tolerability of IMMU-132 Combined with olaparib or talazoparib in athymic nu/nu
nude mice. Mice from the various therapy experiments were treated and weighed periodically
from the time therapy began (i.e., therapy day 0) as indicated in Methods. Weights for the
different treatment groups are represented as the percent of starting body weight (i.e., day 0 =
100%). Toxicity was set at any mouse that dropped to 85% or less. (A) HCC1806 tumor-bearing
mice were treated with olaparib (50 mg/kg; qdx5, repeat weekly x 4 weeks), IMMU-132 (250
g; weekly x 4 weeks), or the combination of both. (B) HCC1806 tumor-bearing mice were
10
treated as above except IMMU-132 was administered at 500 g twice weekly for 4 weeks. (C)
MDA-MB-468 tumor-bearing mice treated with the same doses as in (A) except there was a one
week rest period between the second and third weeks of therapy. (D) HCC1806 tumor-bearing
mice treated with talazoparib (0.33 mg/kg; qdx5 repeated weekly x 4 weeks), IMMU-132 (250
g; weekly x 4 weeks), or the combination of both. Purple arrows indicate administration of
IMMU-132. Green and gray arrows indicate administration of olaparib or talazoparib,
respectively.
11
FANCD2 (166 kDa)
BARD1 (86 kDa)
Rad51 (37 kDa)
ERCC1 (39 kDa)
MDA-MB-231 MDA-MB-468
β-actin (45 kDa)
HCC1806 BT-20
FANCD2 (166 kDa)
BARD1 (86 kDa)
Rad51 (37 kDa)
ERCC1 (39 kDa)
β-actin (45 kDa)
12
Figure S1
13
Figure S2
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Additive
Antagonistic
Synergistic
IMMU-132 (normalized)
Ruc
apar
ib (n
orm
aliz
ed)
MDA-MB-231(BRCA-1/2 w.t.)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Additive
Antagonistic
Synergistic
IMMU-132 (normalized)
Ruc
apar
ib (n
orm
aliz
ed)
MDA-MB-468(BRCA-1/2 w.t.PTEN-mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Additive
Antagonistic
Synergistic
IMMU-132 (normalized)
Tala
zopa
rib (n
orm
aliz
ed)
MDA-MB-231(BRCA-1/2 w.t.)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Additive
Antagonistic
Synergistic
IMMU-132 (normalized)
Tala
zopa
rib (n
orm
aliz
ed)
MDA-MB-468(BRCA-1/2 w.t.PTEN-mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Olap
arib
(nor
mal
ized)
HCC1806(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Olap
arib
(nor
mal
ized)
HCC38(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Olap
arib
(nor
mal
ized)
MDA-MB-468(BRCA-1/2 w.t.PTEN-mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Olap
arib
(nor
mal
ized)
MDA-MB-231(BRCA-1/2 w.t.)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Ruca
parib
(nor
mal
ized)
HCC38(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Ruca
parib
(nor
mal
ized)
HCC1806(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Tala
zopa
rib (n
orm
alize
d)
HCC38(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Tala
zopa
rib (n
orm
alize
d)
HCC1806(BRCA-1/2 mutation)
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Antagonistic
Synergistic
IMMU-132 (normalized)
Olap
arib
(nor
mal
ized)
BT-20(BRCA-1/2 w.t.)
14
Figure S4
0 7 14 21 28 35 42 49 56
90
100
110
120
130
Time Post-Therapy Initiation (days)
Perc
ent o
f Sta
rting
Wei
ght (
mea
n
s.d.
)
0 7 14 21 28 35 42 49 56
90
100
110
120
130
Time Post-Therapy Initiation (days)
Perc
ent o
f Sta
rtin
g W
eigh
t (m
ean
s.
d.)
Olaparib
IMMU-132 + OlaparibIMMU-132
Saline
Figure S3
G1: 81.7 %S: 7.1 %
G2/M: 11.2 %
G1: 75.8 %S: 14.2 %
G2/M: 9.3 %
G1: 49.2 %S: 37.6 %
G2/M: 13.2 %
G1: 79.2 %S: 14.8 %
G2/M: 6.0 %
G1: 55.0 %S: 32.5 %
G2/M: 12.5 %
G1: 38.6 %S: 51.5 %
G2/M: 9.9 %
G1: 31.9 %S: 63.7 %
G2/M: 4.4 %
G1: 82.5 %S: 5.5 %
G2/M: 12.0 %
G1: 82.2 %S: 11.3 %
G2/M: 6.6 %
G1: 67.2 %S: 19.2 %
G2/M: 13.6 %
G1: 66.9 %S: 22.3 %
G2/M: 10.7 %
G1: 42.8 %S: 51.9 %
G2/M: 5.3 %
G1: 93.4 %S: 3.2 %
G2/M: 3.4 %
G1: 85.3 %S: 5.0 %
G2/M: 9.7 %
G1: 76.0 %S: 10.5 %
G2/M: 13.5 %
G1: 43.4 %S: 36.7 %
G2/M: 19.9 %
HCC1806 HCC38 MDA-MB-468 MDA-MB-231
Untreated
Olaparib(10 M)
IMMU-132(5 nM)
IMMU-132+
Olaparib
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0 7 14 21 28 35 42 49 56 63
90
100
110
120
130
Time Post-Therapy Initiation (days)
Perc
ent o
f Sta
rtin
g W
eigh
t (m
ean
s.
d.)
A
C
D
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90
100
110
120
130
Time Post-Therapy Initiation (days)
Perc
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f Sta
rtin
g W
eigh
t (m
ean
s
.d.)
C