SUPPLEMENTAL MATERIALS AND METHODS

Flow cytometry

Different amount of antibodies (0, 0.01, 0.1 and 1µg/106 cells) were incubated with LAN-

1 or activated T cells (ATC) expanded by CD3/CD28 beads for 30 min on ice. After washing, a

secondary PE-conjugated goat anti-human IgG, Fcγ fragment specific (1:100, Jackson

ImmunoResearch) were added for 30 min on ice. Staining was analyzed on a FACSCalibur (BD

Biosciences).

Cell cytotoxicity (51chromium release assay)

Cell cytotoxicity was assayed by 51Cr release as previously described (25). ATCs were

cultured and maintained in F10 medium plus 30 U/ml IL-2, and used at day 14-21. CD3(+) T

cells were >99% by FACS analysis. Normal human primary cells (cardiac myocytes,

hepatocytes, adrenal cortical cells, renal mesangial cells, and pulmonary alveolar epithelial cells)

were purchased from ScienCell Research Laboratories (Carlsbad, CA), and cultured with special

media provided by the manufacturer.

Immunological synapse formation and calcium flux assays

Streptavidin-coated bilayers were generated as described (47) and then incubated with

either biotin-GD2 (0.02 µg/ml) or biotin-huOKT3 IgG1 (1 µg/ml) together with biotin-ICAM-1

(1 µg/ml) (47). Biotin-GD2 was kindly provided by the Consortium for Functional Glycomics,

and biotin-huOKT3 was made by conjugating huOKT3 with Maleimide-PEG2-Biotin (Thermo

Scientific). ATCs were incubated with hu3F8-BsAb or control BsAbs for 30 minutes at 37°C

and then applied to GD2-coated bilayers; or without BsAb for huOKT3 only coated stimulatory

bilayers. After 10 minutes at 37°C, cells were fixed with 2% paraformaldehyde in PBS, washed

1

in PBS, permeabilized with Triton X-100, blocked with BSA, then incubated with mouse anti-

CD3 (BD Pharmingen), followed by staining with Alexa 488-labeled goat anti-mouse antibody

and Alexa 594-phalloidin (Invitrogen) for 1 h. After a final PBS wash, cells were imaged by total

internal reflection fluorescence (TIRF) microscopy (48). Fura-2 Ca2+ imaging of T cell

activation (47) was carried out on either GD2+ICAM-1+ bilayers or on tumor cells. IMR-32

tumor cells were seeded on the imaging chamber at 1x105 cells/well one day prior to the

experiment. Imaging data were analyzed using SlideBook software, Excel (Microsoft) and Prism

(GraphPad Software). Quantification of clearance ratio was performed using two perpendicular

linescans for each cell. For each linescan, the background-corrected mean fluorescence intensity

(MFI) at the edges (positions F1 and F2) of the IS was compared with the background-corrected

MFI of three equally spaced central positions (F3, F4, and F5) as follows: mean(F3 + F4 +

F5)/mean(F1 + F2). Analysis of average Ca2+ flux was performed by calculating the average Fura

ratio of the whole imaging field during the stable plateau phase of the response. Analysis of

single cell Ca2+ flux was performed by normalizing the Fura ratio of each cell using the last

image before the initial rise in Ca2+. Data for conjugate analysis was representative of two

independent experiments.

Additional references

47. Abeyweera TP, Merino E, Huse M. Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells. J Cell Biol 2011;192(4):675-90.

48. Le Floc'h A, Tanaka Y, Bantilan NS, Voisinne G, Altan-Bonnet G, Fukui Y, et al. Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse. J Exp Med 2013;210(12):2721-37.

2

SUPPLEMENTAL FIGURES

A B

C D

Supplemental Figure S1: Tumor free survival in humanized DKO mice. Tumor free survival curves

corresponding to the xenograft tumor growth shown in Figure 5. For sc tumors, mice were sacrificed

when tumor volume reached 2 cm3. For iv tumors, mice either died of tumor growth or were sacrificed

when they developed signs of graft versus host disease. Data were plotted using GraphPad Prism 6

software, and cases were censored when mice were sacrificed or died without tumor. (A) sc tumor plus sc

effector cells (1:1 mixing) model. (B) sc tumor plus iv effector cells model. (C-D) iv tumor plus iv effector

cells model.

3

0 40 80 120 1600.0

0.5

1.0

Days post first treatment

Tum

or fr

ee s

urvi

val Hu3F8-C825

Hu3F8-BsAbPBMC+Hu3F8-C825PBMC+Hu3F8-BsAbATC+Hu3F8-C825ATC+Hu3F8-BsAbFresh T cells+Hu3F8-C825Fresh T cells+Hu3F8-BsAb

0 20 40 600.0

0.5

1.0

Days post first treatment

Tum

or fr

ee s

urvi

val

No treatmenthu3F8-BsAbPBMC+hu3F8-C825PBMC+hu3F8-BsAb

0 40 80 1200.0

0.5

1.0

Days post first treatment

Tum

or fr

ee s

urvi

val

No treatmentATChu3F8-BsAbATC+hu3F8-BsAb

0 40 80 1200.0

0.5

1.0

Days post first treatment

Tum

or fr

ee s

urvi

val

No treatmenthu3F8-BsAbATC+hu3F8-C825ATC+hu3F8-BsAbPBMC+hu3F8-C825PBMC+hu3F8-BsAb

Control PBMC Fresh T cell

NK-depleted PBMC Monocyte-depleted PBMC

Supplemental Figure S2: T cell survival in the sc tumors. Tumors (from groups in Figure 6B)

were harvested 9 days after starting BsAb treatment, and representative images of CD3(+) T cell

IHC staining of tumor sections were shown (200X magnifications).

4

A

50

100

200

400

800

1600

3200

0 10 20 30 40 50 60

Tum

or V

olum

e (m

m3 )

Days post tumor implantation

PBMC

5HLDS(15)BA(Y)

PBMC + 5HLDS(15)BA(Y)

hu3F8-BsAb

PBMC + hu3F8-BsAb

5HLDS(15)BA(Y) injection

hu3F8-BsAb injection

B

50

100

200

400

800

1600

3200

5 12 19 26 33 40 47 54 61 68

Tum

or v

olum

n (m

m3 )

Days post tumor implantation

PBMC

PBMC + 5HLDS(15)BA(Y)

PBMC + hu3F8-BsAb

5HLDS(15)BA(Y) injection

hu3F8-BsAb injection

Supplemental Figure S3: Efficacy of hu3F8-BsAb vs. 5HLDS(15)BA(Y) in humanized DKO mice.

Both panels shown tumor growth of IMR-32 neuroblastoma in sc tumor plus sc effector cells model.

Treatment schedules were indicated in the Figures. (A) 5 millions IMR-32 cells were mixed with 5

millions PBMC cells, and treatment with hu3F8-BsAb (20 ug iv) and 5HLDS(15)BA(Y) (20 ug iv) were

started on Day 4. (B) 5 millions IMR-32 cells were mixed with 10 millions PBMC cells, and treatment

with hu3F8-BsAb (5 ug iv) and 5HLDS(15)BA(Y) (10 ug iv) were started on Day 5. The difference of

the efficacy between hu3F8-BsAb and 5HLDS(15)BA(Y) were even bigger when doses were lowered

down to limited conditions (B).

5

0

20

40

60

80

100

hu3F8-BiTE hu3F8-BsAb hu3F8 Chem Conj

pg/m

l

IL-2

0

30

60

90

120

150

hu3F8-BiTE hu3F8-BsAb hu3F8 Chem Conj

pg/m

l

IL-6

Supplemental Figure S4: Cytokine release from different BsAb formats. Cytokine release from

PBMC in the absence of target tumor cells, comparing hu3F8-BsAb with its equivalent monovalent BiTE

format, and the chemical conjugate of hu3F8-IgG x OKT3.

6