stm.sciencemag.org/cgi/content/full/11/508/eaax8861/DC1

Supplementary Materials for

CD3 bispecific antibody–induced cytokine release is dispensable

for cytotoxic T cell activity

Ji Li, Robert Piskol, Ryan Ybarra, Ying-Jiun J. Chen, Jason Li, Dionysos Slaga, Maria Hristopoulos, Robyn Clark, Zora Modrusan, Klara Totpal, Melissa R. Junttila*, Teemu T. Junttila*

*Corresponding author. Email: junttila.teemu@gene.com (T.T.J.); junttila.melissa@gene.com (M.R.J.)

Published 4 September 2019, Sci. Transl. Med. 11, eaax8861 (2019)

DOI: 10.1126/scitranslmed.aax8861

The PDF file includes:

Fig. S1. Anti-HER2/CD3 treatment-induced cytokine release in mice. Fig. S2. Effect of anti-HER2/CD3 treatment on peripheral T cell status in mice. Fig. S3. Characterization of single-cell RNA-seq data. Fig. S4. Single-cell expression analysis of treatment-based expression patterns within human PBMCs. Fig. S5. Effect of pharmacologic or genetic IL-6 signaling intervention on TDB pharmacology. Fig. S6. Effect of prophylactic IL-1 blockade on anti-HER2/CD3 TDB pharmacology. Fig. S7. CD8-specific expression analysis after TDB treatment. Fig. S8. Expression of TNFR1 and TNFR2 in human T cells and monocytes. Fig. S9. Effect of prophylactic TNF-α blockade on anti-HER2/CD3 TDB pharmacology. Fig. S10. Schematic representation of anti-HER2/CD3 TDB–induced cytokine release.

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/508/eaax8861/DC1)

Data file S1 (Microsoft Excel format). Marker gene sets used for cell type identification in single-cell RNA-seq data. Data file S2 (Microsoft Excel format). Original data.

Figure S1. Anti-HER2/CD3 treatment-induced cytokine release in mice. (A) Non-tumor-bearing

MMTV.huHER2 TG mice were treated with 0.5 mg/kg anti-HER2/CD3 TDB on Day 0 (red). Second dose

was administered 1-28 days later (blue). Systemic cytokine concentrations were detected 2 h after each

dose using Luminex. Bars represent mean SEM. (B) Correlation of systemic and tumoral cytokine

concentrations in vehicle- (black) and TDB- (red) treated mice. MMTV.huHER2 TG mice with palpable

mammary tumors were treated with 0.5 mg/kg anti-HER2/CD3 TDB. Systemic cytokines from serum and

local cytokines from tumor lysate were detected 2 h after TDB dose using Luminex. Tumor lysates were

normalized to 20 mg/ml of total protein concentration before Luminex analysis. (C) Comparison of

reported upregulated cytokines in CD19-CART-treated patients with CRS (1, 3, 5) with cytokines

upregulated in TDB-treated-MMTV.huHER2 TG mice. Mouse cytokine data were compiled from 3

independent experiments. CXCL1 is the murine equivalent of human IL-8. Vehicle in black and TDB-

treated in red. TDB did not induce IL-1. IL-7 and VEGF were undetectable. (D) Tumor-bearing

MMTV.huHER2 (group indicated by “Tumor”) and non-tumor-bearing mice were treated with indicated

doses of anti-HER2/CD3 TDB. Body temperatures (BT, rectal probe) and body weights (BW) were

recorded for 6 days. Data shown as mean± SEM.

Figure S2. Effect of anti-HER2/CD3 treatment on peripheral T cell status in mice. (A-D) Non-tumor-

bearing MMTV.huHER2 TG mice were treated with 0.5 mg/kg anti-HER2/CD3 TDB on Day 0, and

circulating T cells were analyzed by flow cytometry on the indicated days. (A) Peripheral lymphocyte and

T cell numbers were determined by anti-CD45, anti-CD8, and anti-CD4 staining. (B) CD3 expression on T

cell membrane was evaluated by staining with bivalent 2C11. (C) T cell subset analysis. Naïve CD8+ T

cells (CD8+CD62L+CD44-), central memory CD8+ T cells (CD8+CD62L+CD44+), effector memory CD8+ T

cells (CD8+CD62L-CD44+), and effector CD8+ T cells (CD8+CD62L-CD44-). (D) Potential of CD8+ T cells

to respond to chemokines was determined by anti-CXCR3. Data shown as mean SEM. Red = TDB,

black = non-treated (NT). Data shown as mean± SEM. Significant differences compared to NT are

indicated.

Figure S3. Characterization of single-cell RNA-seq data. (A) Schematic illustration of sample

treatment and isolation (w/o: without). Non-treated (gray); 4 h after 1st TDB dose (red); 7 days after 1st

TDB dose (pink); 4 h after 2nd TDB dose (blue). (B) tSNE projection of defined clusters from Fig. 2A, with

individual gene or gene set expression indicated by color intensity.

Figure S4. Single-cell expression analysis of treatment-based expression patterns within human

PBMCs. (A) tSNE projection indicating single cell relationship to treatment. Non-treated (gray); 4 h after

1st TDB dose (red); 7 days after 1st TDB dose (pink); 4 h after 2nd TDB dose (blue), left panel. Cell

numbers of each annotated cell type displayed per treatment, right panel. (B) CD69 expression indicated

on the tSNE projection, left panel, and in the violin plot within CD4 and CD8 cells by treatment, right

panel. Non-treated (gray); 4 h after 1st TDB dose (red); 7 days after 1st TDB dose (pink); 4h after 2nd TDB

dose (blue). (C) IL2RA (CD25) expression indicated on the tSNE projection, left panel, and in the violin

plot within CD4 and CD8 cells by treatment, right panel. Non-treated (gray); 4 h after 1st TDB dose (red);

7 days after 1st TDB dose (pink); 4 h after 2nd TDB dose (blue). (D) Single cell RNA-seq violin plot of

expression across immune cell types of IL1A and IL1RA stratified by treatment. Likelihood-ratio test *

p<1e-10, **p<1e-20, ***p<1e-50, ****p<1e-100.

Figure S5. Effect of pharmacologic or genetic IL-6 signaling intervention on TDB pharmacology.

(A) Dosing and sampling schedule for anti-HER2/CD3 TDB - anti-IL-6 co-treatment of MMTV.huHER2

mice. (B) Impact of 1 g/ml anti-human-IL-6 (clone 6708) and 1 M dexamethasone co-treatment on

TDB-induced in vitro cytokine release using human PBMCs as effector cells. Data shown as mean ± SD.

(C) In vivo effect of anti-IL-6 treatment on anti-HER2/CD3 TDB-induced systemic cytokine release in non-

tumor bearing MMTV.huHER2 transgenic mice. Anti-IL-6 (MP5-20F3, 200 g/animal) was administered

twice, 1 day and 1 h before 0.5 mg/kg anti-HER2/CD3 TDB. Serum was harvested 2 h after TDB dose for

Luminex analysis. (D) In vivo blockade of IL-6 signaling using anti-IL-6 or anti-IL-6R (clone 15A7).

Treatments and sampling as described in (A and C). (E-F) Systemic TDB-induced cytokine release (E)

and lymphocyte margination (F) in IL-6R knockout mice. Treatments and sampling as described in (A and

C). (G) Effect of anti-IL-6 pretreatment on antitumor efficacy of TDB treatment in MMTV.huHER2

transgenic mice with palpable mammary tumors. Data represent tumor volume measurements from

individual animals. Red = TDB, cyan = anti-IL-6, blue = TDB+anti-IL-6, black = vehicle, brown = gD/CD3

TDB (non binding control), magenta = TDB+dexamethasone, Unpaired t-test with Welch’s correction was

used for statistical analysis (F, G). NS: not significant. All experiments in IL-6R KO mice were performed

in female mice using murine ovary and fallopian tube-reactive TDB targeting Ly6/PLAUR domain-

containing protein 1 (LYPD1). Data (C, D, E, F) shown as mean± SEM.

Figure S6. Effect of prophylactic IL-1 blockade on anti-HER2/CD3 TDB pharmacology. (A) Impact of

5 g/ml rIL-1RA (anakinra) co-treatment on TDB-induced in vitro cytokine release using human PBMCs

as effector cells. (B) In vivo effect of anti-IL-1 treatment on anti-HER2/CD3 TDB-induced systemic

cytokine release in non-tumor-bearing MMTV.huHER2 transgenic mice. rIL-1RA-Fc fusion (Genentech,

300 g/animal) was administered twice, 1 day and 1 h before 0.5 mg/kg anti-HER2/CD3 TDB. Serum was

harvested 2 h after TDB dose for Luminex analysis. (C) Influence of 5 g/ml rIL-1RA co-treatment on in

vitro TDB-induced human T cell activation (24 h) and target cell killing after 24 h (1st dose) or 8-day

exposure (2nd dose). Schematic of experimental design is presented in Fig. 1B. (D-F) Effect of rIL-1RA

pretreatment on lymphocyte margination (D; 24 h after TDB dose), tumor-infiltrating lymphocytes (E; 7

days after TDB dose), and antitumor efficacy (F) in MMTV.huHER2 transgenic mice with palpable

mammary tumors. Antitumor efficacy represented as maximum tumor volume change at 14 days (left

panel) and as tumor volume measurements from individual mice (3 right panels). Red = TDB, cyan = rIL-

1RA (anakinra), blue = TDB+ rIL-1RA, black = vehicle. Statistical analysis using unpaired t-test with

Welch’s correction. NS: not significant. Data shown as mean ± SD (A, C) or mean ± SEM (B, D, E, F).

Figure S7. CD8-specific expression analysis after TDB treatment. (A) Schematic illustration of

experimental design and sample isolation. CFSE-labeled SKBR3 cells (green) were incubated with

human CD8+ cells in the presence or absence of TDB for 4 h. CFSE-CD8+ cells were subsequently sorted

and RNA was isolated and sequenced. (B) Volcano plot showing the differential expression statistics

between control (-TDB) and HER2-TDB treated CD8+ cells. Genes significantly (adj. P <0.05) reduced

(≤2-fold) and significantly increased (>2-fold) are highlighted in blue and green, respectively. (C-D)

Volcano plots of differentially expressed genes between control (-TDB) and HER2-TDB-treated CD8+

cells (+TDB), with signature genes annotated in purple for (C)

HALLMARK_TNFA_SIGNALING_VIA_NFKB and (D) HALLMARK_IL6_JAK_STAT3_SIGNALING. (E)

QuSAGE analysis of indicated gene signatures demonstrating differential expression when comparing

control (-TDB) and HER2-TDB-treated CD8+ cells (+TDB). Mean log2-fold change; SD shown. P-values

indicated. Statistical significance was determined using a one-sided test comparing the probability

distribution function of log2-fold changes in a given gene set to a baseline value of zero.

Figure S8. Expression of TNFR1 and TNFR2 in human T cells and monocytes. Expression of TNFRI

(A) and TNFRII (B) was determined by flow cytometry in two-dose human PBMC in vitro model (Fig. 1B).

Figure S9. Effect of prophylactic TNF-α blockade on anti-HER2/CD3 TDB pharmacology. (A) Impact

of 5 µg/ml anti-TNF- (adalimumab) co-treatment on TDB-induced in vitro cytokine release using human

PBMCs as effector cells. (B-C) In vivo effect of anti-TNF-pretreatment on tumor-infiltrating lymphocytes

(B), anti-HER2/CD3 TDB-induced lymphocyte margination (C), and antitumor efficacy (D) was analyzed in

tumor-bearing MMTV.huHER2 transgenic mice. Anti-TNF- (XT3.11, 500 ug/dose/animal) was

administered twice, 1 day and 1 h before 0.5 mg/kg anti-HER2/CD3 TDB. Blood was harvested 24 h after

TDB dose for flow cytometry, and tumors were analyzed 7 days after the TDB dose. Red = TDB, cyan =

anti-TNF-, blue = TDB+ anti-TNF-, black = vehicle. Statistical analysis using unpaired t-test with

Welch’s correction. Data shown as mean SD (A) or mean SEM (B, C).

Figure S10. Schematic representation of anti-HER2/CD3 TDB–induced cytokine release.

T cell triggering Monocyte / macrophage activation

T cell

Tumor

cells

TNF-a

IFN-g

IL-2

Perforin & granzyme

TNF-a

Toxicity

IL-1b

IL-6