www.sciencetranslationalmedicine.org/cgi/content/full/7/287/287ra70/DC1

Supplementary Materials for

Anti-CD20/CD3 T cell–dependent bispecific antibody for the treatment of B cell malignancies

Liping L. Sun,* Diego Ellerman, Mary Mathieu, Maria Hristopoulos, Xiaocheng Chen,

Yijin Li, Xiaojie Yan, Robyn Clark, Arthur Reyes, Eric Stefanich, Elaine Mai, Judy Young, Clarissa Johnson, Mahrukh Huseni, Xinhua Wang, Yvonne Chen, Peiyin Wang, Hong Wang, Noel Dybdal, Yu-Waye Chu, Nicholas Chiorazzi,

Justin M. Scheer, Teemu Junttila, Klara Totpal, Mark S. Dennis, Allen J. Ebens

*Corresponding author. E-mail: sun.laura@gene.com

Published 13 May 2015, Sci. Transl. Med. 7, 287ra70 (2015) DOI: 10.1126/scitranslmed.aaa4802

The PDF file includes:

Fig. S1. Quality control analysis for anti-CD20/CD3 TDB. Fig. S2. Summary of PK analysis for anti-CD20/CD3 TDB in rats. Fig. S3. Representative FACS data for Fig. 1C. Fig. S4. In vitro CD8+ T cell proliferation in the presence of CD20-TDB and BJAB. Fig. S5. Data with three healthy human donors for statistical analysis for Fig. 1 (D to F). Fig. S6. FACS gating strategy for B and T cells with blood and spleen samples of huCD20-huCD3 double-transgenic mice. Fig. S7. Presence of human B and T cells in humanized NSG mice and antigen expression level for human CD20 and CD3. Fig. S8. CD4+ T cell counts in humanized NSG mice upon CD20-TDB treatment. Fig. S9. Control studies with rituximab and rituximab-DANA antibodies. Fig. S10. FACS gating strategy for B and T cells with blood and tissue samples of cynomolgus monkeys. Fig. S11. (A and B) Cytokine production in huCD20-huCD3 double-transgenic mice (A) and in cynomolgus monkeys (B) upon CD20-TDB treatment. Fig. S12. Summary of sample sizes for all presented studies.

Supplementary Materials

Figure S1:

Figure S1: Quality control analysis for antirepresentative lot of CD20-TDB, as well as MS analysis profile (B).

analysis for anti-CD20/CD3 TDB. (A) SEC profile is shown for a TDB, as well as MS analysis profile (B).

CD20/CD3 TDB. (A) SEC profile is shown for a

Figure S2:

Figure S2: Summary of PK analysis for anticoncentration was plotted following a single dose of 0.5 mg/kg or 5 mg/kg rats, where CD20TDB doesn’t bind to rat CD20 and CD3. Cmax, CL and beta halfthe study.

: Summary of PK analysis for anti-CD20/CD3 TDB in rats. CD20-TDB serum concentration was plotted following a single dose of 0.5 mg/kg or 5 mg/kg rats, where CD20TDB doesn’t bind to rat CD20 and CD3. Cmax, CL and beta half-lives were summarized from

TDB serum concentration was plotted following a single dose of 0.5 mg/kg or 5 mg/kg rats, where CD20-

e summarized from

Figure S3:

Figure S3: Representative FACS data for FCD25+.

: Representative FACS data for Fig. 1C: T cell activation characterizedcharacterized as CD69+ and

Figure S4:

Figure S4: In vitro CD8+ T cell proliferation CD8+T cells from human healthy donors were CFSECD20-TDB only, or with CD20-cultured for another 48 hours before analyzed for CFSE intensity by FACS. Twas only observed when CD8+T cells were incubated with BJAB and CD20 TDB together as evidenced by CFSE dilution (in red box).

T cell proliferation in the presence of CD20-TDB and BJABCD8+T cells from human healthy donors were CFSE-labeled and cultured either alone, with

-TDB and BJAB, for 24 hours. Then cells were washed and cultured for another 48 hours before analyzed for CFSE intensity by FACS. T cell proliferation was only observed when CD8+T cells were incubated with BJAB and CD20 TDB together as evidenced by CFSE dilution (in red box).

TDB and BJAB. Purified labeled and cultured either alone, with

TDB and BJAB, for 24 hours. Then cells were washed and cell proliferation

was only observed when CD8+T cells were incubated with BJAB and CD20 TDB together as

Figure S5:

Figure S5: Data with three healthy human donors for statistical analysis for Fighealthy human donors for statistical analysis for Fig.

1 (D to F).

Figure S6:

Figure S6: FACS gating strategy for B and T cells with blood and spleen samples of huCD3 double-transgenic mice.

: FACS gating strategy for B and T cells with blood and spleen samples of

: FACS gating strategy for B and T cells with blood and spleen samples of huCD20-

Figure S7:

Figure S7: Presence of human B and T cells in humanized NSG micelevel for human CD20 and CD3. (A) Shown as representative examples with 2 mice, human CD19+B cells and T cells (CD4+T and CD8+T) were detected by FACS in mouse blood. (B) Human CD3ε and CD20 expression levels on B and T cells from humanized NSG mice were compared to healthy human donor B and T cells by FACS.

: Presence of human B and T cells in humanized NSG mice and antigen expressi. (A) Shown as representative examples with 2 mice, human

CD19+B cells and T cells (CD4+T and CD8+T) were detected by FACS in mouse blood. (B) and CD20 expression levels on B and T cells from humanized NSG mice were

o healthy human donor B and T cells by FACS.

and antigen expression . (A) Shown as representative examples with 2 mice, human

CD19+B cells and T cells (CD4+T and CD8+T) were detected by FACS in mouse blood. (B) and CD20 expression levels on B and T cells from humanized NSG mice were

Figure S8:

Figure S8: CD4+ T cell counts in humanized NSG mice upon CD20-TDB treatment. Human CD34+ cells reconstituted NSG mice (3 per group) were treated intravenously on Day 0 with 3 weekly doses of vehicle or 0.5mg/kg CD20-TDB. Blood was collected at 5 days before treatment (D-5), and on D7, D14, and D21. CD4+T cell counts in blood were determined by FACS. Data is shown as mean and standard deviation.

D-5 D7D14 D21

0.00.20.40.60.80.8

1.3

1.8

2.3

2.8

vehicleCD20 TDB

CD

4+T

cel

l cou

nt (

10^6

)/m

l blo

od

Figure S9:

Figure S9: Control studies with rituximab and rituximabwell as TDB, is potent in killing Bjab cells. (B) RituximabCD20, but has no B cell depletion potency in vivo, as tested in human CD20/CD3 double transgenic mice. Mice were treated with a single dose of vehicle, 10 mg/kg of rituximab, orrituximab-DANA variant. Mouse spleens were harvested 5 days postquantitated by FACS.

rituximab and rituximab-DANA antibodies. (A) Rituximab, as well as TDB, is potent in killing Bjab cells. (B) Rituximab-DANA variant can bind to human CD20, but has no B cell depletion potency in vivo, as tested in human CD20/CD3 double

e were treated with a single dose of vehicle, 10 mg/kg of rituximab, orDANA variant. Mouse spleens were harvested 5 days post-dose, and B cells were

DANA antibodies. (A) Rituximab, as DANA variant can bind to human

CD20, but has no B cell depletion potency in vivo, as tested in human CD20/CD3 double e were treated with a single dose of vehicle, 10 mg/kg of rituximab, or

dose, and B cells were

Figure S10:

Figure S10: FACS gating strategy forcynomolgus monkeys.

: FACS gating strategy for B and T cells with blood and tissue samples of

B and T cells with blood and tissue samples of

Figure S11:

Figure S11: (A and B) Cytokine production in huCD20in cynomolgus monkeys (B) upon CD20CD20-TDB at 0.5 mg/ml, and plasma samples were collected at 2, 8 and 24 hours postCynomolgus monkeys were treated with a single IV dose of CD20samples were collected at 2, 6, and 24 hour postcytokines were measured (n=4 for A, measurement for individual animals

Cytokine production in huCD20-huCD3 double-transgenic mice (A) and in cynomolgus monkeys (B) upon CD20-TDB treatment. Mice were treated with a single dose of

, and plasma samples were collected at 2, 8 and 24 hours postCynomolgus monkeys were treated with a single IV dose of CD20-TDB at 1 mg/kg, and plasma samples were collected at 2, 6, and 24 hour post-dose. Production of a selected group of

for A, error bar shown as mean with STD; n=3 for B, shown as measurement for individual animals).

transgenic mice (A) and TDB treatment. Mice were treated with a single dose of

, and plasma samples were collected at 2, 8 and 24 hours post-dose. TDB at 1 mg/kg, and plasma

d group of ; n=3 for B, shown as

Figure S12:

Figure S12: Summary of sample sizes for all presented studies.

Summary of sample sizes for all presented studies.