1

CD69+ CD107a+ Cytotoxicity

T cell activation

T cell activation Cytotoxicity B

Supplemental Fig. 1: Characteristics of T cell activation and killing induced by HER2-TDB

A

CD

69+

(%

of C

D8+

)

CD

107+

(%

of C

D8+

)

SK

BR

3 ki

lling

(%

)

HER2-TDB (ng/ml) HER2-TDB (ng/ml) HER2-TDB (ng/ml)

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.

2

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.

+20 ng/ml IL-2

CB

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.

3

Supplemental Fig 3:T

um

or

vol (

mm

3 )

No PBMCUntreated

No PBMCHER2-TDB

No PBMCVehicle

PBMC (3)Vehicle

PBMC (3)CTRL-TDB

A B

Day

Individual mouse, drop-outIndividual mouse Treatment group fitted tumor vol

CTRL group fitted tumor vol

A) Activity of HER2-TDB in NOD-SCID mice is dependent on human PBMCs. 5x106 MCF7-neo/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.

4

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.

HumanT cells: EC50 0.4 ng/ml

CD3 TGT cells EC50 2.4 ng/ml

Balb/CT cells

Human T cells

CD3 TG:T cells EC50 11 ng/ml

Balb/C T cells EC50 10 ng/ml

A) In vitro activity of human CD3-specific HER2-TDB

B) In vitro activity of mouse CD3-specific HER2-TDB

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Supplemental Fig 5:

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.

HER2 staining

% change

4D5

CTRL AB

Day

Mea

n tu

mor

vol

ume

(mm

3 +

/- S

EM

)

6

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.

CD3 TGT cells

Balb/CT cells

Human T cells

CD3 TGT cells

Balb/CT cells

B) Murine CD3 expression in CD8+ T cells of CD3-TG mice

A) Human CD3 expression in CD8+ T cells of CD3-TG mice

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Supplemental Fig 6:

Vehicle HER2-TDB

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)

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)

Gated on live cells CD45+ in tumors

CD45+ Leucocytes CD4+ T cells CD8+ T cells

CD69+ (% of CD4+ cells) CD69+ (% of CD8+ cells)

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Supplemental Fig 8:

mPD-1-APC

mPD-1-APC

CD4+ cells

CD8+ cells

mCD45-PE-Cy7

CD4-FITC

CD

8-P

E

B) CT-26-HER2 tumor cells express PD-L1

In vivo tumor cellsCT26/HER2

mPDL-1-PE

CD45 neg cellsisotype

mPDL1

A) CT-26-HER2 tumor infiltrating T cells express PD-1