Supplemental Fig. 1: Characteristics of T cell
activation and killing induced by HER2-TDB
A) T cell activation was detected at various timepoints by staining cells for CD8, CD69 and
CD107a followed by FACS analysis. T cell activation data presented as mean of two
repeats. Effectors CD8+ T cells, target SKBR-3, E:T ratio 3:1. Cytotoxicity was detected
using FACS assay. Effectors CD8+, target SKBR-3, E:T ratio 3:1, Error bar = S.D. in all
panels. (A-C)
B) Cytotoxicity was detected using LDH release assay. Effectors CD8+ T cells, target
BT474, E:T ratio indicated in the figure, time point 19h. T cell activation was measured as in
panel A.
T cell activation
B
Cytotoxicity
SKBR3 killing (%)
CD107+ (% of CD8+)
HER2-TDB (ng/ml)
Cytotoxicity
CD107a+
CD69+
CD69+ (% of CD8+)
A
HER2-TDB (ng/ml)
HER2-TDB (ng/ml)
T cell activation
1
Supplemental Fig 2: Activation of T cells by HER2-TDB
induces T cell proliferation.
A) Proliferation of T cells was measured at day 3 as dilution of CFSE in CD8+/PI- cells with cell
divisions.
B-C) HER2-TDB induces T cell expansion Purified CD8+ T cells were labeled with CFSE
according to manufacturer’s protocol (Invitrogen, #C34554). CFSE-labeled CD8+ T cells were
incubated with target cells in the presence or absence of TDB for 19 hours. T cells were
collected, washed and cultured for 2-7 days (RPMI+10% FBS, +/- 20 ng/ml IL2). Live CD8+
cell number (CD8+/PI-) and the percentage of CFSEdim cells was detected by FACS.
+20 ng/ml IL-2
B
C
2
Supplemental Fig 3:
A) Activity of HER2-TDB in NOD-SCID mice is dependent on human PBMCs. 5x106 MCF7neo/HER2 cells were injected to mice without human PBMC. Mice (N=7) were treated with 0.5
mg/kg i.v. doses of HER2-TDB on days 0, 7 and 14. Tumor volumes from individual mice and
fitted tumor volumes of treatment groups are presented; mice terminated prior to study end are
shown as red traces whereas mice remaining on study to study end are shown as grey traces.
Fitted tumor volume for each treatment group are shown as a solid black line with fitted tumor
volume for comparator control group are shown as a dashed blue line
B) Control TDB which shares the same CD3-arm as HER2-TDB, but has an irrelevant non-binding
target arm has no effect on tumr growth. 5x106 MCF7-neo/HER2 cells were injected together
with 1x107 unstimulated human PBMC. Mice were treated as in panel A.
Individual mouse
Individual mouse, drop-out
No PBMC
Untreated
No PBMC
HER2-TDB
B
No PBMC
Vehicle
PBMC (3)
Vehicle
PBMC (3)
CTRL-TDB
Tumor vol (mm3)
A
Treatment group fitted tumor vol
CTRL group fitted tumor vol
Day
3
Supplemental Fig 4:
TDB mediated killing by CD3-TG splenic T cells. T cells were extracted from
spleens of CD3-TG (magenta), BALB/c mice (blue) or from peripheral blood from
healthy human donors (red). In vitro killing activity of CT26-HER2 cells was tested
using human CD3-specific (UCHT1v9) HER2-TDB (A) or mouse CD3-specific
(2C11) HER2-TDB (B). E:T = 20:1 Assay time: 40 hours. In vitro cytotoxicity was
monitored by flow cytometery.
A) In vitro activity of human CD3-specific HER2-TDB
Human
T cells: EC50 0.4 ng/ml
CD3 TG
T cells EC50 2.4 ng/ml
Balb/C
T cells
B) In vitro activity of mouse CD3-specific HER2-TDB
Balb/C
T cells EC50 10 ng/ml
CD3 TG:
T cells EC50 11 ng/ml
Human
T cells
4
Supplemental Fig 5:
Mean tumor volume
(mm3 +/- SEM)
Effect of 4D5 on established mammary tumors in MMTV-huHER2 transgenic
mice. Mice were treated with single 30 mg/kg IV dose of bivalent murine 4D5
(precursor of humanized 4D5; trastuzumab; N=9) or control mIgG1 antibody (N=8).
4D5 did not significantly affect tumor growth.
% change
CTRL AB
4D5
HER2 staining
Day
5
Supplemental Fig 5:
CD3-TG T cells express both mouse and human CD3 on approximately 50%
level of respective Balb/c mouse or human T cells. T cells were extracted from
spleens of CD3-TG (magenta), BALB/c mice (blue) or from peripheral blood from
healthy human donors (red). stained with (mouse or human) CD8, mouse CD3 (clone
2C11) and human CD3 (cline UCHT1). The figure is gated on CD8+ cells.
A) Human CD3 expression in CD8+ T cells of CD3-TG mice
Human
T cells
CD3 TG
T cells
Balb/C
T cells
B) Murine CD3 expression in CD8+ T cells of CD3-TG mice
Balb/C
T cells
CD3 TG
T cells
6
Supplemental Fig 6:
Anti-tumor activity of HER2-TDB is T cell dependent. 0.1 million CT26-HER2
were injected subcutaneously to BALB/c mice. Mice with established tumors were
treated with vehicle or human CD3-specific HER2-TDB (0.5 mg/kg, qwx3, IV,
n=10)
12−1916 D: TDB−HER2; CT26.WT Her2
Raw Data Only Tumor Volume
0
5
8
2048
Vehicle
●
●
●
1024
10
9
●
●
●
HER2-TDB
●
●
●
●
●
●
●
●
●
●
●
●
●
Tumor Volume ( mm3)
●
512
●
●
●
●
●
●
●
●
256
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
128
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
64
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
32
0
5
10
Day
7
Supplemental Fig 7:
T cells in CT26-HER2 tumors display CD69 activation marker.
0.1 million CT26-HER2 were injected subcutaneously to BALB/c mice. Mice with
established tumors were treated with vehicle, human CD3-specific HER2-TDB or
a CTRL-TDB with irrelevant target arm (0.5 mg/kg, qwx3, IV). Tumors were
charvested 11-35 days after cell injection. Tissues were cut into small pieces and
transferred into gentleMACS C-tubes (Miltenyi, # 130-093-237). Samples were
digested with Collagenase D (1 mg/ml) and DNase I (0.2 mg/ml) in rotating
incubator for 15 minutes then dissociated to achieve single cell suspension. After
anti-CD16/CD32 FcR blocking, cells were stained with the cocktails of surface
markers. (N = 2/group,NT = non treated)
CD45+ in tumors
Gated on live cells
105
104
CD8-PB
CD8+
26.9
10
3
CD4+
2.92
102
0
102
0
103
104
105
5.0
2.5
20
10
hC
B
2.
TD
25
B
B
TD
2.
ER
H
T,
W
G
3T
W
T,
N
T
TD
2.
L.
TD
,N
3TG
hC
D
,H
ER
T
B
0
B
TD
2.
ER
T,
H
W
T,
N
T
W
B
TD
2.
50
D
D
3T
G
,H
ER
,C
TR
L.
TD
,N
T
B
0
75
hC
25
100
,C
TR
50
3TG
W
hC
CD69+ (% of CD8+ cells)
CD8+CD69+ (% of CD8+ cells)
75
3TG
T,
H
D
hC
hC
100
hC
D
ER
B
2.
TD
ER
T,
H
W
T,
N
T
W
D
hC
CD4+CD69+ (% of CD4+ cells)
CD69+ (% of CD4+ cells)
hC
D
W
T,
N
T
0
3TG
D
,N
3TG
T
,C
T
hC
R
L.
D
TD
3TG
B
,H
ER
2.
TD
B
B
2.
TD
ER
T,
H
W
T,
N
T
W
hC
CD8+ T cells
30
0.0
0
D
3TG
D
,N
3TG
T
,C
TR
hC
L.
D
TD
3TG
B
,H
ER
2.
TD
B
7.5
40
3TG
10
CD4+ T cells
hC
D
20
10.0
3TG
D
,N
3TG
T
,C
T
hC
R
L.
D
TD
3TG
B
,H
ER
2.
TD
B
CD4+ (% of CD45+ cells)
CD45+ Leucocytes
30
hC
CD45+ (% of live cells)
40
CD8+ (% of CD45+ cells)
CD4-FITC
Supplemental Fig 8:
A) CT-26-HER2 tumor infiltrating T cells express PD-1
CD8+ cells
100
% of Max
80
60
40
20
1000
10
5
0
0
800
# Cells
600
71.7
400
2
10
10
3
10
2
3
10
4
10
5
PD1-APC
CD8+
26.9
23.1
200
10
4
CD8-PB
10
mPD-1-APC
CD4+
2.92
CD4+ cells
0
0
0
10
2
10
3
10
4
10
5
0
10
2
CD45-PE-Cy7
10
3
10
4
10
100
5
CD4-FITC
CD8-PE
mCD45-PE-Cy7
% of Max
80
60
40
CD4-FITC
20
0
0
10
2
10
3
10
4
10
5
PD1-APC
mPD-1-APC
B) CT-26-HER2 tumor cells express PD-L1
100
1000
80
mPDL1
% of Max
# Cells
In vivo
tumor cells
CT26/HER2
isotype
CD45 neg cells
800
60
600
40
71.7
400
23.1
20
200
0
0
0
10
2
10
3
10
4
CD45-PE-Cy7
10
5
0
10
2
10
3
10
4
10
5
PDL1-PE
mPDL-1-PE
9