Need results section in laymans terms did

TR1801-ADC: a highly potent cMet antibody–drug conjugate with high activity in patient-derived xenograft models of solid tumors Marco Gymnopoulos1 , Oscar Betancourt1, Vincent Blot1, Ryo Fujita1, Diana Galvan1, Vincent Lieuw1, Sophie Nguyen1, Jeanette Snedden1, Christine Stewart1, Jose Villicana1, Jon Wojciak1, Eley Wong1, Raul Pardo2, Neki Patel2, Francois D’Hooge2, Balakumar Vijayakrishnan2, Conor Barry2, John A. Hartley2, Philip W. Howard2, Roland Newman1 and Julia Coronella1

1 Tanabe Research Laboratories U.S.A., Inc., San Diego, CA, USA

2 Spirogen, a member of the AstraZeneca Group, London, UK

Keywords

antibody; drug conjugate; cMet;

gastrointestinal cancer;

pyrrolobenzodiazepine; solid tumors

Correspondence

M. Gymnopoulos, Tanabe Research

Laboratories U.S.A., Inc., 4540 Towne

Centre Court, San Diego, CA 92121, USA

Fax: (858) 558-0650

Tel: (858) 622-7073

E-mail: [email protected]

Marco Gymnopoulos and Oscar Betancourt

contributed equally to the work

(Received 29 May 2019, revised 23 October

2019, accepted 14 November 2019,

available online 3 December 2019)

doi:10.1002/1878-0261.12600

cMet is a well-characterized oncogene that is the target of many drugs

including small molecule and biologic pathway inhibitors, and, more

recently, antibody–drug conjugates (ADCs). However, the clinical benefit

from cMet-targeted therapy has been limited. We developed a novel cMet-

targeted ‘third-generation’ ADC, TR1801-ADC, that was optimized at dif-

ferent levels including specificity, stability, toxin–linker, conjugation site,

and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically

conjugated to the pyrrolobenzodiazepine (PBD) toxin–linker tesirine and

has picomolar activity in cancer cell lines derived from different solid

tumors including lung, colorectal, and gastric cancers. The potency of our

cMet ADC is independent of MET gene copy number, and its antitumor

activity was high not only in high cMet-expressing cell lines but also in

medium-to-low cMet cell lines (40 000–90 000 cMet/cell) in which a cMet

ADC with tubulin inhibitor payload was considerably less potent. In vivo

xenografts with low–medium cMet expression were also very responsive to

TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor

showed a substantially reduced efficacy. Furthermore, TR1801-ADC had

excellent efficacy with significant antitumor activity in 90% of tested

patient-derived xenograft models of gastric, colorectal, and head and neck

cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of

10 head and neck cancer models showed complete tumor regression after a

single-dose administration. Altogether, TR1801-ADC is a new generation

cMet ADC with best-in-class preclinical efficacy and good tolerability in

rats.

Abbreviations

ADC, antibody–drug conjugate; DAR, drug–antibody ratio; ERK, extracellular signal-regulated kinase-1; HGF, hepatocyte growth factor; HIC,

hydrophobic interaction chromatography; kD, kilo Dalton; MAPK, mitogen-activated protein kinase; MMAE, monomethyl auristatin E; NAC,

N-acetyl cysteine; PBD, pyrrolobenzodiazepine; PDX, patient-derived xenograft; PI3K, phosphatidylinositol 3-kinase; PK, pharmacokinetics;

SEC, size-exclusion chromatography; TFF, tangential flow filtration; TGI, tumor growth inhibition; TMA, tissue microarray; vc, valine–

citrulline; VEGF, vascular endothelial growth factor.

54 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Antibody–drug conjugates (ADCs) with over 40 years

of research are a promising and fast-growing class of tar-

geted anticancer immunotherapies. These agents com-

bine the specificity of antibodies with the potency of

chemotherapeutics by attaching highly cytotoxic pay-

load–linkers covalently to monoclonal antibodies

(Mukherjee et al., 2019). With four approved ADCs in

the United States for hematological cancers and solid

tumors and over 60 ADCs at various stages of develop-

ment, the field is rapidly expanding and evolving (Beck

et al., 2017). The shortcomings of older generation

ADCs, mainly serum stability and low tolerability in

humans, could be vastly improved by changing and opti-

mizing conjugation chemistry, antibody–drug ratio, and

using a new repertoire of linker–toxins (tubulin inhibi-

tors, pyrrolobenzodiazepines (PBDs), irinotecan deriva-

tives, and DNA monoalkylators) (Agatsuma, 2017;

Beck et al., 2017; Mantaj et al., 2017; Tolcher, 2016).

The proto-oncogene MET encodes the receptor tyr-

osine kinase (cMet). Upon binding of its ligand, hepa-

tocyte growth factor (HGF), a series of intracellular

signals are initiated involving morphogenic differentia-

tion, wound healing, motility, invasion, and antiapop-

tosis (Petrini, 2015; Zhang et al., 2018). Aberrant cMet

expression or constitutive activation of the cMet sig-

naling pathway due to amplification, overexpression of

its ligand HGF, and mutation in MET is seen in many

human tumor types and is the rationale for developing

cMet-targeting therapeutics (Gherardi et al., 2012).

Many small molecule cMet inhibitors and pathway-in-

hibiting biologics were developed over the last decade

with limited or no clinical success (Puccini et al.,

2019). The therapeutics that were successful seem to be

limited to small subsets of patients with MET-ampli-

fied cancers (Comoglio et al., 2018). Novel cMet-tar-

geting therapies, such as ADCs, are in development,

which are independent of MET amplification status

and target any cMet-overexpressing cancer (Wang

et al., 2017; Yang et al., 2019).

TR1801-ADC is an innovative new generation ADC

with highly optimized features, including a DNA-dam-

aging payload, distinguishing itself from other cMet ther-

apeutics such as ADCs with tubulin inhibitor payload.

2. Materials and methods

2.1. Cell lines and culture conditions

SNU-1 (RRID:CVCL_0099), SNU-16 (CVCL_0076),

SNU-5 (CVCL_0078), NCI-H1373 (CVCL_1465),

NCI-H1975 (CVCL_1511), NCI-H1573 (CVCL_1478),

NCI-H441 (CVCL_1561), SW1417 (CVCL_1717), SW-

480 (CVCL_0546), NCI-H747 (1587), HCT-116

(CVCL_0291), Detroit 562 (CVCL_1171), and FaDu

(CVCL_1218) were purchased from the American Tis-

sue Type Collection (ATCC, Manassas, VA, USA).

MKN-45 (CVCL_0434) was obtained from Deutsche

Sammlung von Mikroorganismen and Zellkulturen

(DSMZ, Braunschweig, Germany) and SNU-620

(CVCL_5079) from the Korean Cell Line Bank (KCLB,

Seoul, Korea). All cell lines were authenticated at

ATCC by analyzing short terminal repeats and found

to be correct matches. All experiments were performed

with mycoplasma-free cells. Cell lines were maintained

according to the cell bank’s recommendations or in

normal growth medium, RPMI-1640 (Thermo Fisher,

#21870092, Waltham, MA, USA) with 2 mM glutamine

(Thermo Fisher, #25030164) and 10% FBS (Thermo

Fisher, #26140079) at 37 °C and 5% CO2.

2.2. P3D12 cMet antibody humanization

Five different methods were used to humanize the

P3D12 anti-cMet antibody: CDR grafting, grafting of

abbreviated CDRs, SDR transfer, Frankenstein

approach, and veneering. The abbreviated CDR

method and the SDR method yielded the same amino

acid sequence. The resulting four heavy chain and four

light chain variable regions were cloned into the

pFUSE hIgG2 and pFUSE hj vectors, respectively,

giving 16 possible combinations of heavy/light chain

pairs, which were expressed in Expi293 cells. Antibod-

ies were purified, affinities determined, and biophysical

characteristics assessed.

2.3. Subclass switching and site-specific cysteine

incorporation

The variable regions of the mouse P3D12 antibody

were cloned into vectors containing the nucleotide

sequence of the constant regions of human IgG1 and

human IgG2. The site-specific cysteines were intro-

duced utilizing nearby restrictions sites and Gibson

assembly.

2.4. Antibody expression and conjugation

The humanized, IgG2 monoclonal antibody, hD12,

was engineered to incorporate an unpaired cysteine

residue on each heavy chain Fc region to generate

TR1801-Ab, which was produced from a stably trans-

fected Chinese hamster ovary cell line and purified

using protein A affinity chromatography followed by

55Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

size-exclusion chromatography (SEC). Prior to conju-

gation, the hD12 antibody was partially reduced using

495 molar equivalents of L-glutathione (Sigma-

Aldrich, #G6529, St. Louis, MO, USA), and after

incubating for 1 h at room temperature, the glu-

tathione was removed by tangential flow filtration

(TFF). For conjugation, 10 molar equivalents of a

10 mM SG3249 DMSO solution were added to the

reduced antibody, and the reaction continued for 1 h

at room temperature. The reaction was quenched with

the addition of a 15-fold excess of N-acetyl cysteine

(NAC; Acros Organics, #160280250, The Hague,

Netherlands) and underwent incubation at room tem-

perature for another 15 min. The unreacted NAC-

capped SG3249 was removed using TFF while buffer

exchanging into 25 mM histidine, 85 g�L�1 trehalose

dihydrate, pH 5.5 buffer. The ADCs, TR1801-ADC,

was recovered and sterile-filtered using 0.2-µM PES fil-

ter, and polysorbate-80 (Amresco, # M126, Solon,

OH, USA) was added up to a final concentration of

0.02% v/v. The overall product recovery was 87% for

the conjugation reaction.

2.5. Analytical methods

The concentration of TR1801-ADC was determined by

subtracting the ratio of absorbance values measured at

280 and 330 nm (A280/A330) for SG3249 from the A280

measurement for TR1801-ADC to account for the

absorbance of the conjugated payload. The remainder

was divided by the molar extinction coefficient (e280) of the hD12 antibody. To determine the monomer content

and weighted average of drug-to-antibody ratio (DAR)

values, the TR1801-ADC sample was diluted to

1 mg�mL�1 and analyzed using analytical SEC and

hydrophobic interaction chromatography (HIC). SEC

analysis of TR1801-ADC using a Tosoh Bioscience

TSKgel SuperSW mAb column and mobile phase buffer

containing 200 mM potassium phosphate pH 6.95,

250 mM potassium chloride, and 10% isopropanol (v/v)

demonstrated ~ 3% high molecular weight species. No

low molecular weight species were observed (Fig. 1).

The weighted average of DAR value was determined

using a HIC Butyl-NP5 column equilibrated with

mobile phase buffer A (1.5 M ammonium sulfate, 25 mM

sodium phosphate, pH 6.50). After sample loading and

washing, a mobile phase B (25 mM sodium phosphate,

pH 6.50, 25% v/v isopropanol) gradient was applied to

sequentially elute the low-DAR to high-DAR species

(Fig. 1). Based on this HIC method, the TR1801-ADC

weighted average of DAR was determined to be 1.96

and < 1 percent of the material was unconjugated

(Table S1). Proteolytic digestion (FabRICATOR

enzyme; Genovis, Lund, Sweden) of TR1801-ADC fol-

lowed by reversed-phase chromatography (A330 detec-

tion) indicated that > 74% of SG3249 was conjugated

to the Fc fragment, and peptide mapping showed the

peptide containing the engineered cysteine reside was

conjugated to SG2349 and no other SG3249-conjugated

peptides were identified (data not shown).

2.6. Met degradation and phospho-Erk ELISA

Total cMet in SNU-16 cells was measured with the SEC-

TOR Imager 2400 (MSD, Gaithersburg, MD, USA).

Cells were treated with anti-cMet antibodies and incu-

bated for 24 h. Extracellular signal-regulated kinase-1

(ERK) phosphorylation in MKN-45 cells was measured

with the SECTOR Imager 2400. MKN-45 cells were

incubated with cMet antibodies for 15 min. All assays

were performed as recommended by the manufacturers.

2.7. Cytotoxicity assays

Cell viability was determined by measuring the lumi-

nescence after adding the CellTiter-Glo� 2.0 reagent

(Promega, #G9242, Madison, WI, USA). Cancer cells

were seeded overnight in growth media and incubated

at 37 °C, 5% CO2, and 95% humidity. ADCs or the

PBD warhead, SG3199, were added in serial dilutions

starting with concentrations of 100 nM for ADCs and

10 nM for free drug. Cells were exposed to test articles

for 5 days. IC50s were calculated by nonlinear regres-

sion using sigmoidal curve fitting in PRISM 7 (Graph-

Pad, San Diego, CA, USA).

2.8. In vivo tumor xenograft studies in mice

All in vivo xenograft studies were approved by the

IACUC of Tanabe Research Laboratories, USA, Inc.

(San Diego, CA, USA) and performed according to

the company’s Institutional Animal Care Guidelines.

H1975 and H1373 cancer cell lines were implanted

subcutaneously at 5 9 106 cells/animal into the right

flank of female Nu/Nu mice obtained from Charles

River (Wilmington, MA, USA). Animals were ran-

domized after the average tumor volume reached 200– 300 mm3. Mice were given a single intravenous injec-

tion of ADC, nontargeting control ADC, or vehicle

control at doses described in the Figs 1–4. Body

weight and tumor volume were measured 2–3 times

per week over the entire duration of the studies.

Tumor volume was calculated as follows: V

(mm3) = 0.5236 9 length (mm) 9 width2 (mm).

Tumor volumes � SEM were plotted in PRISM 7

(GraphPad). Statistical significance was determined

56 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.

with a one-way ANOVA with Tukey’s or Dunnett’s

multiple comparison test dependent on whether groups

were compared to a control group or not. When only

two dose groups were compared, an unpaired two-

tailed t-test was performed in PRISM 7.

2.9. Rat pharmacology study

Male Sprague Dawley rats were given an intravenous

bolus injection of 0.5, 1, 1.5, and 2 mg�kg�1 of ADCs.

Body weights and general clinical observations were

recorded daily over the entire 21 days of the study.

Blood samples for pharmacokinetics (PK) were drawn

predose, 4, 24, 48, 96, 168, 336, and 504 h after injec-

tion of test articles and collected in heparin-coated

tubes, followed by 14 000 g centrifugation for 5 min.

Plasma concentrations of ADCs were measured by

ELISA. Noncompartmental pharmacokinetic parame-

ters were calculated using WinNonlin software (Phar-

sight, Mountain View, CA, USA).

2.10. TR1801-ADC Intact ELISA (ADC) and Total

Antibody ELISA (TAB)

For the ADC ELISA, a high-affinity, anti-payload-

specific monoclonal antibody was used to capture

TR1801-ADC in serum samples and an anti-human

FC-specific secondary antibody was used for detection.

This assay format allowed for the detection of

TR1801-ADC species containing either 1 or 2 conju-

gated payloads but did not detect unconjugated

TR1801-ADC species. For the TAB ELISA, a purified

extracellular domain of cMet was used as the coating

reagent to capture TR1801-ADC in serum samples via

antibody–antigen interactions. The immobilized

TR1801-ADC species were detected using an anti-hu-

man secondary antibody specific for kappa light

chains. Since site-specific conjugation of the payload

to the attachment site on the heavy chain did not

affect antigen binding, this assay format demonstrated

equivalent detection of both payload-conjugated and

Fig. 1. Analytical characterization of TR1801-ADC. (A) SEC analysis of TR1801-ADC using a Tosoh Bioscience TSKgel SuperSW mAb column

and 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride, and 10% isopropanol (v/v) mobile phase. Note: The peak

absorbance at ~ 22.5 min is from the formulation buffer. (B) HIC analysis of TR1801-ADC using a Butyl-NP5 column equilibrated with mobile

phase buffer A (1.5 M ammonium sulfate, 25 mM sodium phosphate, pH 6.5) and linear elution gradient elution to mobile phase B (25 mM

sodium phosphate, pH 6.50, 25% v/v isopropanol).

57Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

unconjugated TR1801-ADC species. For the TAB

ELISA, the lower limit of quantification (LLOQ) in

rat serum is 15 ng�mL�1 and the minimum required

dilution (MRD) is 10-fold, while for the ADC ELISA,

the LLOQ is 25 ng�mL�1 and the MRD is 100-fold.

Comparison of the individual TR1801-ADC serum

concentrations and/or relevant toxicokinetic parame-

ters (e.g., Cmax and AUC) determined using the ADC

and TAB ELISAs could provide insights into potential

deconjugation of the payload after dosing. The TAB-

to-ADC ratio of ~ 1 would indicate unconjugated

TR1801-ADC species were not detected, and the

observed TR1801-ADC toxicokinetic properties were

consistent between assays. A TAB-to-ADC ratio

demonstrably > 1 would suggest the presence of

unconjugated TR1801-ADC species and potential loss

of the payload.

2.11. IHC of tissue microarrays (TMAs) and PDX

sections

TMAs (US Biomax, Derwood, MD, USA, or US Bio-

labs, Rockville, MD, USA) or patient-derived xeno-

graft (PDX) cancer tissue sections (CrownBio, Beijing,

China) were treated at 100 °C in EDTA buffer pH 9

for 20 min for antigen unmasking. Primary rabbit

monoclonal antibody cMet SP44 (Abcam, #ab227637,

Cambridge, MA, USA) (diluted 1 : 200) or rabbit IgG

Fig. 2. In vitro potency and in vivo efficacy of TR1801-ADC with lung cancer cell lines H1975 (60 000 cMet receptors/cell) and H1373 (97 000

cMet receptors/cell) with medium–low cMet expression. Five-day CellTiter-Glo� cytotoxicity assays were run as duplicates and repeated at least

one time. Lung cancer xenografts in Nu/Nu mice were inoculated with 5 9 106 cells/mouse, and mice were injected with test articles at an

average tumor volume of 200–300 mm3. Tumor volume is plotted in mm3 � SEM. (A) Lung cancer cell lines H1975 and H1373 were treated with

TR1801-ADC, nontargeting ADC secukinumab–SG3249, cMet-vc-MMAE (10-point dilution series with a starting concentration of 100 nM), or free

PBD toxin SG3199 (starting concentration of 10 nM). (B) Lung cancer xenografts H1975 and H1373 were treated with single intravenous doses of

vehicle (19 PBS), TR1801-ADC (1 and 0.5 mg�kg�1), cMet-vc-MMAE (5 and 1 mg�kg�1), and nontargeting ADC (1 mg�kg�1) with eight animals

per group. Statistics: one-way ANOVA with Dunnett’s multiple comparison test. Shown is only the significance between cMet-vc-MMAE,

TR1801-ADC, and rituximab-SSC-SG3249 in comparison with control (*P < 0.05, **P < 0.01, ***P < 0.001).

58 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.

isotype control (1 : 200) (Abcam, #ab27478) were

incubated with tissue sections for 60 min at room tem-

perature. Goat anti-rabbit IgG- horseradish peroxidase

conjugate was used as a detection antibody (Leica

Biosystems, #DS9800, Wetzlar, Germany) at

25 µg�mL�1 concentration for 60 min at room temper-

ature. All stained TMAs were scanned with the Nano-

Zoomer Image system� (Hamamatsu, Hamamatsu

City, Japan), and IHC staining intensity was scored

according to following formula: Total score = (% at

0) 9 0 + (% at 1) 9 1 + (% at 2) 9 2 + (% at 3) 9 3

with 0 = no staining, 1 = weak staining, 2 = medium

staining, and 3 = strong staining.

2.12. PDX cancer models

cMet ADCs were evaluated in HuPrime� cancer PDX

models (CrownBio) in female BALB/c nude mice (14– 15 weeks old). Each mouse was subcutaneously inocu-

lated at the right flank with a 2–3 mm (diameter)

tumor piece of one of the tested PDX models. Mice

were randomly grouped into six groups (n = 10 ani-

mals) according to the tumor size average of 200 mm3.

A single dose (otherwise it is indicated when more

than one dose was given) of test articles was adminis-

tered intravenously into the tail vein at the dose con-

centrations indicated. Animals were checked daily for

morbidity and mortality. Tumor size was measured

twice a week with calipers. Tumor volume was calcu-

lated using the formula TV = 0.5 9 A 9 B2. PRISM 7

(GraphPad) was used to perform a one-way ANOVA

and Dunnett’s multiple comparison analysis to calcu-

late P values. TGI% was calculated as follows: TGI%-

= (mean (control day 9)-mean (control day 0))-(mean

(test article day 9)-mean (test article day 0)/ (mean

(control day 9)- mean (control day 0)*100.

2.13. PDX 3D ex vivo experiments

Single-cell suspensions were isolated from HuPrime� cancer PDX models (CrownBio) from tumors that

reached a volume of 500–800 mm3. 2 9 105 cells were

mixed with 1% methylcellulose and seeded into a 96-

well plate. Plates were incubated overnight at 37 °C with 5% CO2 and 95% humidity. Test articles were

added and incubated for 7 days. Cell viability was

determined by adding CellTiter-Glo� reagent (Pro-

mega, #G7572) and reading luminescence on an EnVi-

sion Multilabel Reader (PerkinElmer, Waltham, MA,

USA). Data were displayed in PRISM 7 (GraphPad),

and IC50s were calculated by using a nonlinear regres-

sion model with sigmoidal fitting.

3. RESULTS

3.1. Humanization of P3D12 antibody and

subclass switching to reduce agonist activity and

potential immunogenicity

The mouse, anti-human cMet antibody P3D12 was

selected as the lead antibody because of its high-affin-

ity binding to humans and cynomolgus monkey

(0.8 nM kD), and rat cMet (15.6 nM kD) (Table S2),

which allowed to investigate the tolerability of ADCs

in another species besides nonhuman primates. By

switching the subclass from IgG1 to IgG2, the agonist

activity was significantly reduced (~ 50%) to minimize

mitogen-activated protein kinase (MAPK) and phos-

phatidylinositol 3-kinase (PI3K) pathway activation

(Fig. S1). The humanization of P3D12 (hD12) had no

negative impact on affinity (0.26 nM kD for human

cMet, 7 nM kD for rat cMet), internalization, or

Fig. 3. Expression of cMet in patient samples of gastric, colon, biliary tract, and head and neck (H&N) cancers. IHC was performed with

SP44 rabbit monoclonal cMet antibody on TMAs (80–100 cores per indication were analyzed). Intensity of cMet staining was scored on a

scale from 0 to 300 (H-score) and grouped in four levels of Met expression (none, low, medium, and high) and plotted as % of patient

samples.

59Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

nonagonist activity compared to the parental mouse

antibody (Fig. S1, Table S2).

3.2. Five cleavable PBD linker–toxins stochastically conjugated to wt hD12 showed

different activities in vitro and in vivo

To choose the optimal linker–toxin for our cMet

ADC, TR1801-ADC, with respect to activity and tol-

erability, five cleavable (Val-Ala) PBD linkers from

Spirogen Ltd (London, UK). were conjugated and

evaluated using the wt hD12 cMet antibody (rat and

cynomolgus monkey cross-reactive) for their activity

in vitro and in vivo, and tolerability in rats (Fig. S2).

hD12-SG3259 exhibited the highest activity

(IC50 = 24.4 pM and 86% maximal killing) in the

H1975 (60 000 cMet/cell) lung cancer cell line followed

by hD12-SG3246 (IC50 = 91 pM, 62%), hD12-SG3315

(IC50 = 146 pM, 46%), and hD12-SG3249

(IC50 = 153 pM, 79%; Table S3, Fig. S2A). The hD12-

SG3227 ADC was the least active (IC50 = 410 pM,

77%). The difference in potencies was less prominent

in another cMet-expressing lung cancer cell line H1373

(97 000 cMet/cell) with IC50 of 11 pM (SG3246),

20 pM (SG3315), 22 pM (SG3249), 28 pM (SG3227),

and 31 pM (SG3259) (Fig. S2A, Table S3). The differ-

ences in potency seen in vitro with H1975 cells

matched the efficacy seen in the H1975 subcutaneous

Fig. 4. Preclinical assessment of TR1801-ADC in 10 HuPrime� gastric cancer PDX models. Female BALB/c nude mice were treated with a

single intravenous dose of vehicle control, TR1801-ADC, or nontargeting ADC (secukinumab–SG3249) when subcutaneous tumors reached

an average size of 200 mm3. Ex vivo 3D methylcellulose assays were performed on selected gastric PDX over a 7-day period with TR1801-

ADC, free PBD toxin SG3199, and cisplatin. Nine-point dilution series were prepared with starting concentrations of 50, 10, and 100 µM,

respectively. Assay was run in triplicates with an n = 1. (A) Two representative gastric PDX models GA3121 and GA0152. Tumor growth of

each group (n = 10) was monitored after a single intravenous administration of vehicle (19 PBS), TR1801-ADC (1, 0.5, 0.25, and

0.125 mg�kg�1), or nontargeting ADC (1 mg�kg�1). Statistics: one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05,

**P < 0.01, ***P < 0.001). (B) Ex vivo 3D assay performed with GA3121 and GA0152 PDX models and treated with free PBD toxin

(SG3199), TR1801-ADC, or cisplatin. (C) Representative IHC staining with rabbit monoclonal cMet antibody (SP44) on tissue sections of

gastric cancer PDX models GA3121 and GA0152. (D) Plot of 10 gastric cancer PDX models. Tumor growth inhibition (%) at different dose

concentrations (1, 0.5, 0.25, and 0.125 mg�kg�1) of TR1801-ADC.

60 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.

mouse xenograft model (Fig. S2B). All ADC variants

had significant antitumor activity compared to PBS

control (P < 0.0001) in all dose groups (Table S4).

While single intravenous high doses (0.5 mg�kg�1) of

cMet ADC variants lead to full tumor regression in all

groups with no significant differences, the lower dose

groups (0.125 mg�kg�1) differed in antitumor activity.

hD12-SG3259 was significantly more potent at the low

dose than SG3315 (P < 0.0001) and SG3227

(P < 0.0001); but not in comparison with SG3249 and

SG3246 (Table S4). In general, hD12-SG3259, hD12-

SG3249, and hD12-SG3246 were the most potent,

while hD12-SG3227 and hD12-SG3315 were the least

potent. Based on these data, SG3249 and SG3259 were

chosen as the lead PBD toxin–linker candidates and

further investigated in a rat tolerability study. SG3259-

conjugated cMet ADC (hD12-SG3259) was less well

tolerated than hD12-SG3249. Animals were found

moribund or dead after 7 or 10 days in the high-dose

groups of hD12-SG3259, and rapid body weight loss

over 20% was observed after 7 days. hD12-SG3249

ADC was well tolerated in all dose group with no sig-

nificant body weight loss or any other clinical observa-

tions (Fig. S2C). Thus, SG3249 (tesirine) was selected

as lead PBD toxin–linker for all further studies.

3.3. Site-specific conjugation of SG3249 to hD12

produced a stable and homogeneous ADC

In general, site-specific conjugates are more homoge-

nous drugs and have several benefits over stochastically

linked toxin–linkers. The stability of site-specific ADCs

is highly increased, which often leads to improved tol-

erability and a better PK profile in vivo (Strop et al.,

2015; Strop et al., 2013). The heavy chain constant

region of hD12 was used, and tesirine was conjugated

site-specifically to introduced cysteines in the CH2

domain. The product was a homogeneous ADC (97%

monomeric) with a DAR of 2 (average DAR 1.96)

(Fig. 1, Table S1). Site-specific hD12 SG3249 conjugate

was slightly more potent in vitro than stochastically

conjugated hD12-SG3249 (32 versus 153 pM)

(Table S3). Both ADCs showed similar and significant

in vivo antitumor activity at both dose levels compared

to a control (P < 0.0001) (Fig. S3A and S3B). The cir-

culating terminal half-life for TR1801-ADC was

14 days in rats (Fig. S3C). Comparing the serum con-

centrations measured using a Total Antibody PK

Assay, which detects both conjugated and unconju-

gated antibody species, and an ADC PK Assay, which

detects only conjugated species, significant deconjuga-

tion of payload–linker was not observed. The tolerabil-

ity in rats was good with continuous body weight gain

in all dose groups and no remarkable clinical observa-

tions (Fig. S3D). The site-specific cMet hD12–tesirine conjugate was named TR1801-ADC.

3.4. TR1801-ADC was potent in 14 cMet-

expressing cancer cell lines and two xenografts

with medium–low cMet expression

Fifteen cancer cell lines from different organs (gastric,

colorectal, and head and neck and lung cancers) were

tested for sensitivity to TR1801-ADC. The cMet

expression levels ranged from zero (SNU-1), low

(SNU-16), medium (H1373) to high (MKN-45). IC50s

varied between 4 pM (H441) and 13 nM (H1573)

(Table 1).

Table 1. Potency and efficacy of TR1801-ADC in 15 cancer cell line with various cMet expression levels and MET amplification status.

Cancer cell lines were exposed to TR1801-ADC for 5 days before CellTiter-Glo� reagent was added. IC50s and % maximum killing were

determined in GRAPHPAD PRISM 7 after sigmoidal curve fitting of dose–response curves.

Cell line Cancer type MET copy number (CCLE) MET receptor # TR1801-ADC IC50 (pM) SD % max kill SD n

SNU-5 Gastric 3 460 000 15.6 2.7 97.5 0.4 2

MKN-45 Gastric 12 295 000 15.8 6.7 97.8 1.6 4

SNU-620 Gastric 45 294 000 197.9 67.3 99.3 0.3 3

H1373 Lung 3 97 000 2272.7 857.0 95.5 1.2 6

H441 Lung 3 74 000 4.2 1.4 95.2 3.1 2

H1573 Lung 21 73 000 13 444.8 20 146.4 88.8 2.3 3

H1975 Lung 3 60 000 346.4 192.1 97.6 1.7 5

Detroit 562 Head and neck 4 59 000 11 030.5 7547.0 68.3 2.5 2

H747 Colorectal 2 52 000 3230.0 192.3 99.1 0.1 2

SW1417 Colorectal 3 38 000 3494.0 1887.9 92.9 2.7 4

SNU-16 Gastric 3 37 000 4664.0 478.0 90.5 6.2 2

HCT116 Colorectal 2 37 000 190.2 58.1 98.9 0.9 3

FaDu Head and neck 2 34 000 327.5 4.7 98.1 0.0 2

SW480 Colorectal 2 5000 1380.0 90.5 92.4 10.3 2

SNU-1 Gastric 2 0 24 373.7 3677.2 97.4 2.0 3

61Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

Two lung cancer cell lines H1975 and H1373 with

medium–low cMet expression and no MET gene

amplification (60 000 cMet/cell and 97 000 cMet/cell)

are sensitive to TR1801-ADC, with IC50s of 320 pM

(98% maximal killing), with H1975 of 2.2 nM (96%),

and with H1373 cancer cell lines in cytotoxicity

assays (Table S3). A cMet ADC, based on stochasti-

cally conjugated cleavable monomethyl auristatin E

[cMet-valine–citrulline (vc)-MMAE], showed low

activity (IC50 = 68 nM and > 100 nM with 52% and

29% maximal killing in H1975 and H1373). This

was comparable to nontargeting secukinumab– SG3249 (IC50 = 26 nM and 48 nM and 83% or 68%

maximal killing; Fig. 2A, Table S3). This difference

in activity was also seen in the corresponding xeno-

grafts (Fig. 2B) and was significant using an

unpaired two-tailed t-test comparing dose groups of

the H1975 study (P = 0.0029, high-dose groups;

P = 0.023, low-dose groups) and in the low-dose

group of the H1373 model (P < 0.0001; Table S5).

Overall, TR1801-ADC (at 0.5 and 1 mg�kg�1) and

cMet-vc-MMAE (at 5 mg�kg�1) showed significant

antitumor activity in both models in comparison

with the PBS control (Table S5). However, cMet-vc-

MMAE, even though dosed five times or two times

higher (5 and 1 mg�kg�1) than TR1801-ADC, did

not cause full tumor regression in either model.

TR1801-ADC showed complete tumor regression in

the highest dose (1 mg�kg�1) and partial tumor

regression in the low dose (0.5 mg�kg�1). The

response was not only more pronounced with

TR1801-ADC but also more durable with no tumor

regrowth over 100 days in the 1 mg�kg�1 group

(Fig. 2B).

3.5. cMet is highly expressed in cancers of the

gastrointestinal tract and head and neck cancers

Met is highly expressed in several solid tumor types,

for example, lung, renal cell, esophageal, and others

(Kim et al., 2017; Pyo et al., 2016; Sweeney et al.,

2002; Tsao et al., 1998; Xu et al., 2015). We stained

TMAs of four different cancer indications of interest

(gastric, colon, biliary, and head and neck cancers)

and quantified cMet expression. cMet expression was

seen in all four indications, but the largest patient

sample number with high cMet expression (H-

score > 150) was seen in Western population samples

of gastric cancer (55%), colon cancer (30%), and head

& neck (20%) and biliary cancers (13%) (Fig. 3).

Based on these results, gastric, colorectal, and head

and neck cancers were selected for translational PDX

studies.

3.6. GI cancer PDX models are highly sensitive

to TR1801-ADC in vivo and ex vivo

To further substantiate the antitumor activity of TR1801-

ADC inmore translatable in vivomodels, 10 gastric cancer

PDX models were chosen with different expression levels

of cMet. Two representative models GA3121 and GA0152

are shown with high (H-score 295) and medium–high (H-

score 173) cMet expression (Fig. 4C). A durable complete

response was seen in both subcutaneous models at 1 and

0.5 mg�kg�1 using a single intravenous dose of TR1801-

ADC. Partial responses were seen with single low doses of

TR1801-ADC at 0.25 and 0.125 mg�kg�1 (Fig. 4A). In

comparison, nontargeting secukinumab–SG3249 had only

a minor effect on tumor growth. The in vivo response to

TR1801-ADC correlated well with the response ex vivo.

TR1801-ADCwas slightly less potent in the GA0152 PDX

model (medium–high cMet) in vivo and had a higher IC50

ex vivo of 1.7 nM (98% maximal killing) in comparison

with 12 pM (99%) in the GA3121 model (high cMet;

Fig. 4B). The free toxin SG3199 had similar IC50s in both

PDX models of 29 pM (100%) and 36 pM (99%) for

GA3121 and GA0152. The control test article cisplatin

was less potent with an IC50 in the 3–4 digit nM range

(366 nM for GA3121 and 1460 nM for GA0152). Signifi-

cant tumor growth inhibition (TGI) was seen in all models

at 1 and 0.5 mg�kg�1 ranging from > 100% to 40%

(Fig. 4D, Table S6). Seven of 10 gastric PDX showed com-

plete tumor regression at 1 mg�kg�1, and 5/10 models, at

0.5 mg�kg�1.

We also wanted to test whether the high expression of

cMet in colon cancer translates into a high antitumor

activity of our cMet ADC in colorectal PDX models. As

anticipated, TR1801-ADC was highly active in colorec-

tal cancer PDX models with statistically significant

growth inhibition in 9/10 PDX models (Table S6). Com-

plete tumor regression was observed in 40% (4/10) when

treated with 1 mg�kg�1 of ADC. The other 5/10 colorec-

tal PDX models showed partial tumor regression, and

one model showed no significant antitumor response

(Fig. 5D, Table S6). The PDX model CR3150 with high

cMet expression (H-score = 300) was more responsive

to a single intravenous dose of TR1801-ADC than the

model CR0126 with medium–high more heterogeneous

cMet expression (H-score = 190) (Fig. 5A,C). The non-

targeting control secukinumab–SG3249 had some effect

in model CR3150 on tumor growth but on the same

level as the four times lower dose of TR1801-ADC. The

corresponding ex vivo experiments showed that the free

toxin SG3199 had a 26-fold lower potency in model

CR0126 (IC50 = 500 pM, 100% maximal killing) in

comparison with CR3150 (IC50 = 18 pM, 94%) and

TR1801-ADC was 11 times less potent in CR0126

62 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.

(IC50 = 10.54 nM, 87%) than model CR3150

(IC50 = 0.97 nM, 99%) (Fig. 5B). The cisplatin control

was less potent with IC50s > 1000 nM in both models.

Altogether, TR1801-ADC was highly active in gas-

trointestinal cancers with a stable and robust antitu-

mor response.

3.7. TR1801-ADC was active in head and neck

PDX models with medium-to-high cMet

expression

Eight of 10 head and neck PDX models with var-

ious levels of cMet expression showed significant

growth inhibition in subcutaneous xenografts

(Table S6). Complete tumor regression was

observed in 30% (3/10) of models when treated

with a single dose of 1 mg�kg�1 TR1801-ADC.

50% (5/10) of the models showed partial regres-

sion, and two models showed no significant anti-

tumor activity (Fig. 6B, Table S6). The three

models with complete tumor regression had cMet

expression ranging from high (HN3533 H-

score = 300), medium–high (HN0696 H-

score = 180), to medium (HN0635 H-score = 130;

Fig. 6A). Model HN0635 with medium cMet

responded best to TR1801-ADC treatment.

Fig. 5. Preclinical assessment of TR1801-ADC in 10 HuPrime� colorectal cancer PDX models. Female BALB/c nude mice were treated with

a single dose of vehicle control, TR1801-ADC, or nontargeting ADC (secukinumab–SG3249) when subcutaneous tumors reached an average

size of 200 mm3. Ex vivo 3D methylcellulose assays were performed on selected colorectal PDX over a 7-day period with TR1801-ADC,

free PBD toxin SG3199, and cisplatin. Nine-point dilution series were prepared with starting concentrations of 50, 10, and 100 µM,

respectively. Assay was run in triplicates with n = 1. (A) Two representative colorectal PDX models CR3150 and CR0126. Tumor growth of

each group (n = 10) was monitored after a single intravenous administration of vehicle (19 PBS), TR1801-ADC (1, 0.5, 0.25, and

0.125 mg�kg�1), or nontargeting ADC (1 mg�kg�1). Statistics: one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05,

**P < 0.01, ***P < 0.001). (B) Ex vivo 3D assay performed with CR3150 and CR0126 PDX models and treated with free PBD toxin

(SG3199), TR1801-ADC, or cisplatin. (C) Representative IHC staining with rabbit monoclonal cMet antibody (SP44) on tissue sections of

colorectal cancer PDX models CR3150 and CR0126. (D) Plot of 10 colorectal cancer PDX models. TGI% at different dose concentrations (1,

0.5, 0.25, and 0.125 mg�kg�1) of TR1801-ADC.

63Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

4. Discussion

The HGF receptor, cMet, is highly expressed in vari-

ous solid tumor indications (Giordano et al., 1992;

Isaksson-Mettavainio et al., 2008; Sierra and Tsao,

2011; Yap et al., 2011), and mutations in the MET

gene (Pilotto et al., 2017) (e.g., exon 14 splice variant:

NSCLC 3–10%, gastric 7.1%) and gene amplifications

are relatively rare (Jardim et al., 2015) (6% gastric

cancer, 1% lung cancer). Low normal tissue expression

of cMet is seen in the GI tract, liver, skin, and lung,

and can increase upon tissue repair and regeneration

(Jung et al., 2012; Prat et al., 1991).

cMet expression in tumors is associated with poor

prognosis and resistance to targeted therapy, for exam-

ple, epidermal growth factor receptor and vascular

endothelial growth factor pathway inhibitors (Bean

et al., 2007; Zhang et al., 2003). There has been a

major effort to develop small molecule cMet pathway

inhibitors (crizotinib, cabozantinib, capmatinib, tepo-

tinib, and glesatinib) and inhibitory antibodies (onar-

tuzumab, ficlatuzumab, emibetuzumab, SAIT-301, and

ABT700) (Lee et al., 2018a; Puccini et al., 2019).

Targeting cMet with small molecule inhibitors and

nonagonistic antibodies has not been very successful in

the clinic. These therapeutics are limited to MET-am-

plified or, more precisely, subsets of cancers with con-

stitutively activated Met pathway. The omission or

difficulty of patient stratification contributed to many

failed clinical trials (Hughes and Siemann, 2018, 2019).

These setbacks opened up opportunities for other tar-

geted therapies that are independent of MET amplifi-

cation and Met pathway activity such as ADCs (Wang

et al., 2017; Yang et al., 2019). The antibody used in

TR1801-ADC is a nonagonistic binder that enhances

the safety of our ADC by reducing downstream signal-

ing of cMet (PI3K/AKT, ERK/MAPK and SRC/focal

adhesion kinase pathways) and potential activation of

tumor-promoting events (Greenall et al., 2012; Organ

and Tsao, 2011). We decided to use the PBD payload

tesirine (SG3249) on TR1801-ADC, which showed the

best compromise between potency and tolerability in

our studies. Tesirine is currently in preclinical develop-

ment for several solid tumors and hematological can-

cers (Cho et al., 2018; Hartley et al., 2018; Tiberghien

et al., 2016) and in clinical trials (Horwitz et al., 2017;

Fig. 6. Preclinical assessment of TR1801-ADC in 10 HuPrime� head and neck cancer PDX models. Female BALB/c nude mice (n = 10 per

group) were treated with vehicle control, TR1801-ADC, or nontargeting ADC (secukinumab–SG3249) when subcutaneous tumors reached

an average size of 200 mm3. (A) Three representative head and neck PDX models HN3533 (H-score = 300), HN0696 (H-score = 180), and

HN0635 (H-score = 130) and corresponding IHC with rabbit monoclonal cMet antibody (SP44). Tumor growth of each group (n = 10) was

monitored after a single intravenous administration of vehicle (19 PBS), TR1801-ADC (1, 0.5, 0.25, and 0.125 mg�kg�1), or nontargeting

ADC (1 mg�kg�1). Statistics: one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Plot of 10

head and neck cancer PDX models. TGI% at different dose concentrations (1, 0.5, 0.25, and 0.125 mg�kg�1) of TR1801-ADC.

64 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.

Rudin et al., 2017). Tesirine is a newer generation

PBD (Tiberghien et al., 2016) with reduced hydropho-

bicity and same potency as talirine (SGD-1910).

Increasing hydrophilicity of the payload leads poten-

tially to a better tolerated toxin by reducing off-target

toxicity (Lucas et al., 2018; Nakada et al., 2019). The

site-specific conjugation of SG3249 to engineered cys-

teines on the constant region of the heavy chain led to

a homogeneous ADC with high stability and long

half-life in rats. It was well tolerated in rats with no

major clinical findings or pronounced weight loss even

though cMet might be expressed in some normal tis-

sues of the rat (liver, lung, GI tract). More extensive

pharmacological studies, especially in nonhuman pri-

mates, will have to clarify to which extent normal

cMet expression will contribute to on-target toxicity.

Based on preclinical and clinical studies with tesirine

as payload (Cho et al., 2018; Rudin et al., 2017; Uda-

gawa et al., 2019), off-target toxicity will most likely

be the critical factor that will determine the maximum

tolerated dose in animals and humans.

TR1801-ADC was potent and highly efficacious in

in vitro and in vivo experiments with medium–low cMet expression cancer cell lines (H1975 and H1373)

in which a cMet ADC with MMAE payload was less

active even at higher dose concentrations. We theorize

that the receptor number was the driver of activity in

this case and not the sensitivity of the cancer to the

mechanism of action. This demonstrates that PBD

payloads clearly outperform less potent tubulin inhibi-

tor payloads in medium–low-expressing tumor models.

Any concern that the higher potency could cause a

lower tolerability in animals was unsubstantiated as

TR1801-ADC, which is cross-reactive to rat cMet, was

well tolerated in a rat pharmacology study.

Taxanes in general and ADCs with tubulin inhibitor

payloads (e.g., trastuzumab-DM1) had low activity in

tumors of the gastrointestinal tract as shown in several

clinical trials and meta-analysis (McClelland et al.,

2009; Quiles et al., 2010; Shi et al., 2017; Swanton

et al., 2009; Swanton et al., 2006). We can assume that

cMet ADCs with tubulin inhibitor toxins would share

the same fate despite the high and abundant cMet

expression in GI cancers and TR1801-ADC takes

advantage of this opportunity. We were able to repro-

duce published data that showed high and abundant

expression of cMet in GI cancers (Gayyed et al., 2015;

Lee et al., 2018b; Paliga et al., 2017; Safaie Qamsari

et al., 2017; Wu et al., 2014; Yildiz et al., 2016) by per-

forming IHC on TMAs. Ex vivo experiments revealed

a high activity of TR1801-ADC in gastric and colorec-

tal PDX models. The sensitivity of TR1801-ADC was

dependent on the activity of the PBD warhead

(SG3199) in the tested PDX ex vivo models. Reasons

for the differences in PBD sensitivity in the PDX mod-

els we examined are unknown; however, recently pub-

lished work (Hartley et al., 2018) may indicate

important factors. For example, it was shown that cer-

tain defects in the DNA repair protein excision repair

cross-complementation group 1 or homologous recom-

bination repair can sensitize cancer cells to the toxin– linker warhead SG3199 of tesirine. In the opposite

case, expression of the multidrug resistance gene 1 (P-

gp) could lower the sensitivity to SG3199 (Hartley

et al., 2018).

Based on the encouraging ex vivo results, we tested the

in vivo activity of TR1801-ADC in gastric, colorectal,

and head and neck PDX models with cMet expression

between H-scores 130 and 300, the assumed range for

robust antitumor activity of TR1801-ADC. Significant

antitumor activity was seen in 90% of all PDX models

tested, and full tumor regression was seen in the majority

of gastric cancer PDX models and a significant number

of colorectal and head and neck cancer models. There

was no clear dependency between cMet expression and

response to TR1801-ADC other than that some amount

of cMet must be expressed to make the ADC efficacious.

A thorough analysis of molecular factors in the tested

PDXmodels that could modulate sensitivity as described

recently for SG3199 would be of great interest and could

assist to stratify future cMet-positive patients that will

benefit the most from TR1801-ADC (Hartley et al.,

2018; Hughes and Siemann, 2019).

5. Conclusions

These data show that TR1801-ADC could become a

best in class therapeutic for the treatment of cMet-

overexpressing tumors and can target tumors with

even low cMet expression. Currently, TR1801-ADC is

being assessed in a phase 1 clinical trial for cMet-over-

expressing solid tumors.

Acknowledgements

We thank Eduardo Padlan for the support of the

humanization of our P3D12 cMet antibody.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

MG designed the studies. OB, RF, DG, VL, SN, CS,

JV, JW, and EW contributed to the execution of the

65Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

M. Gymnopoulos et al. A novel cMet ADC with activity in solid tumor PDX models

experiments. MG and OB performed the analysis and

interpretation of the data. NP, FD, BV, and CB pro-

duced the ADC and performed analytical testing. MG

and OB drafted the manuscript. VB, NP, JH, PH,

RN, and JC reviewed the manuscript.

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Supporting information

Additional supporting information may be found

online in the Supporting Information section at the end

of the article. Fig. S1. Nonagonist activity and in vitro degradation

of humanized IgG2 cMet antibody hD12.

Fig. S2. PBD toxin linker assessment in vitro and

in vivo on cMet hD12 antibody.

Fig. S3. In vitro and in vivo assessment of site-specific

cMet hD12-SSC-SG3249 in comparison to stochastic

hD12-SG3249.

Table S1. TR1801-ADC quality attributes.

Table S2. Affinity and species cross-reactivity of mouse

and humanized cMet P3D12 antibody clone.

Table S3. Cytotoxicity of TR1801-ADC, cMet ADC

variants and control ADCs with H1975 and H1373

cancer cell lines.

Table S4. Significance of antitumor activity of hD12

PBD drug-linker variants in a H1975 xenograft model.

Table S5. Significance of antitumor activity of

TR1801-ADC at different concentrations and stochas-

tically coupled cMet-vc-MMAE ADC in H1975 and

H1373 xenograft models.

Table S6. Significance of antitumor activity of

TR1801-ADC in PDX models.

68 Molecular Oncology 14 (2020) 54–68 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A novel cMet ADC with activity in solid tumor PDX models M. Gymnopoulos et al.