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The preclinical profile of the duocarmycin-based HER2-targeting ADC SYD985

predicts for clinical benefit in low HER2-expressing breast cancers.

Miranda M.C. van der Lee(1), Patrick G. Groothuis(1), Ruud Ubink(1), Monique A.J. van

der Vleuten(1), Tanja A. van Achterberg(1), Eline M. Loosveld(1), Désirée Damming(1),

Daniëlle C.H. Jacobs(1), Myrthe Rouwette(1), David F. Egging(1), Diels van den

Dobbelsteen(1), Patrick H. Beusker(2), Peter Goedings(3), Gijs F.M. Verheijden(4),

Jacques M. Lemmens(4), Marco Timmers(4), Wim H.A. Dokter(1)

Authors’ Affiliations: Departments of (1)Preclinical; (2)Medicinal & Protein Chemistry;

(3)Medical R&D; (4)New Molecular Entities, Synthon Biopharmaceuticals, Nijmegen, the

Netherlands.

Running title: SYD985 is effective in low HER2-expressing breast cancer.

Corresponding Author: Wim Dokter, Synthon Biopharmaceuticals , Microweg 22,

6503 GN Nijmegen, the Netherlands. Tel: +31 (0)24 372 7700, email:

Wim.Dokter@Synthon.Com

Disclosure of potential conflict of interest: All authors are employees of Synthon

Biopharmaceuticals BV. JL owns stock of Synthon BV.

Financial Support of this study came from Synthon Biopharmaceuticals BV.

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Supplementary Tables.

Table S1. Summary of status of tumors used in cell line and patient-derived xenograft

models.

Model Type HER2 FISH HER2 IHC ER/PR

BT474 CDX + 3+ +

MAXF 1162 PDX + 3+ -

ST313 PDX - 2+ +

HBCx-34 PDX - 2+ +

MAXF 449 PDX - 1+ -

MAXF MX1 PDX - 1+ -

HBCx-10 PDX - 1+ -

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Table S2. SYD985 and T-DM1 IC50, 95% confidence interval and % efficacy values

from the in vitro cytotoxicity assays as depicted in Figure 1B.

Cell line HER2 level

SYD985 T-DM1

IC50 (ng/ml) 95% CI %

efficacyIC50

(ng/ml) 95% CI % efficacy

SK-BR-3 3+ 6.9 6.5 to 7.3 99 15.7 14.7 to 16.9 98

UACC-893 3+ 54.1 49.1 to 59.6 93 35.9 33.6 to 38.3 88

NCI-N87 3+ 24.5 23.0 to 26.0 94 44.9 41.3 to 48.7 91

SK-OV-3 2+ 32.4 30.2 to 34.8 86 112 66.4 to 189 72

MDA-MB-175-VII 1+ 67.4 37.4 to 121 94 314 201 to 490 93

ZR-75-1 1+ 14.9 10.8 to 20.6 61 >1000 89

SW-620 0 >1000 34 >1000 29

NCI-H520 0 >1000 66 >1000 81

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Table S3. Pharmacokinetic parameters for ADC (SYD985 and T-DM1) in BT-474-

tumor-bearing mice after a single i.v. bolus injection of ADC.

ADC Analyte dose (mg/kg)

t ½(hr)

Cmax

(ug/mL)C0

(ug/mL)AUClast

(hr*ug/mL)CL

(mL/hr/kg)Vss

(mL/kg)

T-DM1 ADC 1 67 15 16 527 1.8 120

ADC 5 141 99 106 2970 1.7 198

SYD985 ADC 1 9.5 17 17 54.9 18.2 92.9

ADC 3 55 50 59 151 19.7 201.8

Supplementary Figure Legends.

Figure S1. In vitro cytotoxicity. Comparison of two batches of T-DM1. Four tumor cell

lines were treated for 6 days with T-DM1 EU batch N0001B02 or US batch 535405.

Induction of cytotoxicity was quantified by measuring the percentage of cell survival

Figure S2. A-G HER2 expression in BT-474 (A.) and breast cancer patient derived

xenografts (B-G). Tumors were stained with the primary Ab anti-HER2/neu (4B5) rabbit

monoclonal on a Discovery automated platform (Ventana-Roche).

Figure S3. HER2 cell surface expression quantified on a panel of 8 cell lines using the

DAKO Qifikit. HER2 binding sites were saturated with mouse anti-human HER2

MAB1129.

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Figure S4. In vitro cytotoxicity. A) Induction of cytotoxicity by seco-DUBA quantified

after 6 days for the NCI-N87 and UACC-893 cells and after 12 days for the MDA-MB-

175-VII and ZR-75-1 cells. B) Lack of trastuzumab cytotoxic response on the UACC-

893 cells treated for 6 days with trastuzumab and SYD985. C) Intact ADC (SYD985

conjugated antibody) concentrations of 100 µg/ml SYD985 after 96 hrs incubation at

37°C in human plasma with increasing concentrations of recombinant mouse

carboxylesterase 1c (mCES1c) (± SEM, n=2). D) SYD985 and T-DM1 lack cytotoxic

activity on HER2-negative NCI-H520 cells measured after 6 days of treatment.

Figure S5. In vitro bystander killing. (A) Cytotoxicity induced on HER2-positive, HER2-

negative and co-cultured cells after 12 days of treatment with 1 g/ml of ADCs or 10 nM

of active toxin using the CellTiter-Glo luminescence assay. Percentage survival was

calculated related to untreated cells quantified by measuring the percentage of cell

survival.

Figure S6. Mean ADC (SYD985 conjugated antibody) concentrations in plasma in

carboxylesterase CES1c knock-out (-/-) , CES1c heterozygeous (+/-) and CES1c wild

type (+/+) mice, after single i.v. bolus injection of SYD985 at 5 mg/kg (+/- SEM, n = 3).

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Figure S1.

0.1 1 10 100 1000 100000

20

40

60

80

100

120

SK-BR-3

SK-BR-3

Concentration (ng/ml)

% S

urvi

val

SW-620

SW-620

MDA-MB-175-VII

MDA-MB-175-VII

SK-OV-3

SK-OV-3

EU batch N0001B02 on:

US batch 535405 on:

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Figure S2.

A. B.

D.C.

BT-474 HER2 3+ MAXF 1162 HER2 3+

ST313 HP HER2 2+ HBCx-34 HP HER2 2+

E. MAXF 449 TNBC HER2 1+ F. MAXF MX1 TNBC HER2 1+ HBCx-10 TNBC HER2 1+G.

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Figure S3.H

ER2

bind

ing

site

s pe

r cel

l

SK-BR-3

UACC893

NCI-N87

SK-OV-3

ZR-75-1

MDA-MB-17

5-VII

SW62

0

NCI-H52

00

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

900,000

1,000,000

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Figure S4.

A.

-13 -12 -11 -10 -9 -8 -70

20

40

60

80

100

120

NCI-N87

Log conc (molar drug equivalents)

% S

urvi

val

ZR-75-1MDA-MB-175UACC893

0.1 1 10 100 1000 100000

20

40

60

80

100

120

Concentration (ng/ml)%

Sur

viva

l

trastuzumabSYD985

B.

D. HER2-negative NCI-520 cells

-13 -12 -11 -10 -9 -8 -7 -60

20

40

60

80

100

120

SYD985

Isotype control ADC

T-DM-1Seco-DUBA

Log conc (molar drug equivalents)

% s

urvi

val

C.

Con

cent

ratio

n (µ

g/m

l)

0

20

40

60

80

100

120

µg/ml mCES1c0 10 100 200 400

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Figure S5.

0102030405060708090

100110120130140150160

% S

urvi

val

MDA-MB-175-VII / NCI-H520 12 days

HER2 1+MDA-MB-175-VII

HER2 0NCI-H520

Co-cultureMDA-MB-175-VII

+NCI-H520

SYD985

Isotype control ADC

T-DM-1Seco-DUBA

SYD985 T-DM1 Isotype control ADC Seco-DUBA0

20

40

60

80

100

120

% S

urvi

val

100% + 0%80% + 20%60% + 40%40% + 60%20% + 80%0% + 100%

Co-cultured cells:SK-BR-3 + NCI-H520 HER23+ HER20

A.

B.

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Figure S6.

Time (h)

Con

cent

ratio

n (µ

g/m

L)

0 100 200 3000.01

0.1

1

10

100

1000

CES1c +/+CES1c +/-CES1c -/-

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