Supplementary Figure 1

Supplementary Figure 1. TWEAK is internalized and degraded following binding to Fn14-positive cells. A, Serum-starved human U118 glioma cells plated on coverslips were either left untreated (0 min) or treated (100 ng/ml) with FLAG-tagged soluble TWEAK (Alexis Inc.). Unbound FLAG-TWEAK was washed away and 30 min later the cells were fixed in 3% buffered formalin. Indirect immunofluorescence analysis was performed using an anti-FLAG antibody (Sigma) or control mouse IgG (eBioscience) followed by incubation with a goat anti-mouse IgG F(ab’)2 Alexa Fluor 488 secondary antibody conjugate (Molecular Probes). Photographs were taken using a fluorescent microscope. Magnification is 100X. B, Myc-tagged soluble TWEAK was purified from a stably-transfected HEK-293T cell line and added (100 ng/ml) to human endothelial cell cultures at 4 °C for 30 min. Unbound myc-TWEAK was washed away and the cells were warmed to 37 °C to allow ligand internalization. The cells were harvested at the indicated times by trypsinization and equivalent amounts of protein were subjected to SDS-PAGE and Western blot analysis using anti-myc and anti-actin antibodies.

NT 30 min 30 min

anti-FLAG anti-FLAG control IgG

A

+ FLAG-TWEAK

Myc

BTime Since Washing Away

Unbound Myc-TWEAK

Time, sec

0 100 200 300 400

RU

0

10

20

30

TWEAK

6.25 nM12.5 nM

25 nM50 nM

Kd=3 nM

Time, sec

0 100 200 300 400R

U

0

100

200

GrB-TWEAK

6.25 nM

12.5 nM25 nM50 nM

Kd=8 nM

A

Supplementary Figure 2

B

Supplementary Figure 2. Surface plasmon resonance analysis of TWEAK, GrB-TWEAK and GrB-Fc-IT4 binding to recombinant Fn14 extracellular domain. Sensograms showing binding of TWEAK (A), GrB-TWEAK (A) and GrB-Fc-IT4 (B) to Fn14 immobilized on a CM5 chip. GrB did not bind to immobilized Fn14.

Time, sec

0 100 200 300 400 500

RU

0

1000

2000

300050 nM

Kd=18 nM

GrB-Fc-IT4

25 nM

12.5 nM

6.25 nM

Supplementary Figure 3

MDA-MB-231Non-treated

GrB-TWEAK,100 nM, no 1°Ab

GrB-TWEAK50 nM

GrB-TWEAK100 nM

Supplementary Figure 3. GrB-TWEAK specifically internalizes into Fn14-expressing MDA-MB-231 cells. Cells were untreated or treated with 50 or 100 nmol/L GrB-TWEAK for 5 hours. The cells were fixed, acid washed to remove surface-bound material, permeabilized, and immunostained for the presence of GrB (green). The cells were counterstained with propidium iodide (red) to identify nuclei and visualized using a confocal (Zeiss LSM 510) microscope.

0 0.2 0.4 0.6 0.8 1.00

0.2

0.4

0.6

0.8

1.0

5-FU

Normalised Isobologram

GrB

-TW

EA

K

1:38400 1:7680 1:1536 1:307.21:61.44

12

3

4

5

0 0.2 0.4 0.6 0.8 1.00

0.2

0.4

0.6

0.8

1.0

Cisplatin

Normalised Isobologram

GrB

-TW

EA

K

1:3332 1:666.4 1:133.28 1:26.656

1

234

0 0.2 0.4 0.6 0.8 1.00

0.2

0.4

0.6

0.8

1.0

GemZAR

Normalised Isobologram

GrB

-TW

EA

K

1:12680 1:2536 1:507.2 1:101.441:20.288 1:4.0576 1:0.81152

1234

5

6

7

0 0.3 0.6 0.9 1.20

0.5

1.0

1.5

2.0

Doxorubicin

Normalised Isobologram

GrB

-TW

EA

K

1:1724 1:344.8 1:68.96 1:13.7921:2.758 1:0.552

123

4

5

6

Supplementary Figure 4. Effect of GrB-TWEAK in combination with 5-FU, Cisplatin, GemZAR and Doxorubicin on MDA-MB-231 cells. Cells (2,000 cells/well) were seeded in 96-well plates and treated with ~ IC25 doses of GrB-TWEAK (10 nM for MDA-MB-231) and with various concentrations of each chemotherapeutic agent as described in Supplementary Materials and Methods. After 72 h, cell viability was assessed. Normalized isobolograms were then generated using the CalcuSyn software, depicting CI values of combination drug studies. CI < 1, CI = 1, and CI > 1 indicate synergism, additive interaction, and antagonism, respectively. No difference in sensitivity to GrB-TWEAK was observed based on the order of treatment. Data shown is from pre-treatment of cells with chemotherapeutic agents prior to addition of GrB-TWEAK. All experiments were performed in triplicate.

Supplementary Figure 4

Supplementary Figure 5

Supplementary Figure 5. Caspase-independent PARP cleavage. Pre-incubation of MDA-MB-231 and HT-29 cells with z-VAD-fmk (50 μM) for 1 h prior to 100 nM GrB-TWEAK treatment for 2 hours did not fully block PARP cleavage.

50 µM z-VAD-fmk

100 nM GrB-TWEAK

-- + -- + -- + -- +

-- -- + + -- -- + +

PARP

β-actin

MDA-MB-231 HT-29

Cleaved PARP

PI-9

Fn14

β-actin

MD

A-MB-4

35

AAB-5

27T-

24M

DA-M

B-231

HT-

29H

T-10

80SKO

V3M

E-180

N-8

7PC

-3U

87-M

GW

M35

Cal

u-3

eB1

PI-9(longer exposure)

Fn14

β-actin

U87

N87 HT-

29M

DA-M

B-231

AsPc1

Cap

an-1

Cap

an-2

L3.6

P1H

CC

827

A549

H35

8H

CC

1703

H20

73H

1975

PI-9(shorter exposure)

Supplementary Figure 6

Supplementary Figure 6. Expression of the GrB inhibitor proteinase inhibitor 9 (PI-9) in various cancer cell lines. Equivalent amounts of whole cell lysate were subjected to SDS-PAGE and Western blot analysis using anti-Fn14, PI-9 and β-actin antibodies.

Supplementary Figure 7

0 5 10 15 20 25 30-5

0

5

10

15

20

25

GrB-TWEAK (40 mg/kg)

GrB-TWEAK (20 mg/kg)

GrB (40 mg/kg)Control (Saline)

% B

od

y W

eig

ht

Ch

an

ge

S

EM

Days Post Tumor Implantation

A

B

0 10 20 30 40 50

0

10

20

30

Control (Saline)

GrB-TWEAK (40 mg/kg)

GrB-Fc-IT4 (20 mg/kg)

GrB-Fc-IT4 (40 mg/kg)

% B

od

y W

eig

ht

Ch

an

ge

S

EM

Days Post Tumor Implantation

Supplementary Figure 7. Percent change in body weight of each group of mice from the xenograft experiments, plotted as a function of time. A. GrB or GrB-TWEAK was administered (i.v.) to mice bearing HT-29 xenograft tumors. B. Administration of GrB-TWEAK or GrB-Fc-IT4 into mice bearing MDA-MB-231/Luc breast tumor orthotopic xenografts. Arrows indicate treatment days

Supplementary Figure 8

Supplementary Figure 8. Schematic of GrB-Fc-IT4 fusion protein. GrB-Fc-IT4 is a homodimer in which an Fc domain of human IgG1 is covalently linked to GrB (N-terminus) and the anti-Fn14 humanized scFv of ITEM-4 (C-terminus). Also shown are the disulfide bridge of hinge and the approximate position of the N-linked oligosaccharides attached at Asn297 in the IgG1 Fc-domain.