89Zr-Onartuzumab PET imaging of c-MET receptor dynamics
89Zr-Onartuzumab PET imaging of c-MET receptor dynamics
Martin Pool 0 1 2
Anton G. T. Terwisscha van Scheltinga 0 1 2
Arjan Kol 0 1 2
Danique Giesen 0 1 2
Elisabeth G. E. de Vries 0 1 2
Marjolijn N. Lub-de Hooge 0 1 2
0 Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen , Groningen , The Netherlands
1 Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen , P.O. Box 30.001, 9700 RB Groningen , The Netherlands
2 Department of Medical Oncology, University Medical Center Groningen, University of Groningen , Groningen , The Netherlands
Purpose c-MET and its ligand hepatocyte growth factor are often dysregulated in human cancers. Dynamic changes in cMET expression occur and might predict drug efficacy or emergence of resistance. Noninvasive visualization of cMET dynamics could therefore potentially guide c-METdirected therapies. We investigated the feasibility of 89Zr-labelled one-armed c-MET antibody onartuzumab PET for detecting relevant changes in c-MET levels induced by c-METmediated epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib resistance or heat shock protein-90 (HSP90) inhibitor NVP-AUY-922 treatment in human nonsmall-cell lung cancer (NSCLC) xenografts. Methods In vitro membrane c-MET levels were determined by flow cytometry. HCC827ErlRes, an erlotinib-resistant clone with c-MET upregulation, was generated from the exon-19 EGFR-mutant human NSCLC cell line HCC827. Mice bearing HCC827 and HCC827ErlRes tumours in opposite flanks underwent 89Zr-onartuzumab PET scans. The
Onartuzumab; c-MET; HSP90; 89Zr; Erlotinib; PET
HCC827-xenografted mice underwent 89Zr-onartuzumab
PET scans before treatment and while receiving biweekly
intraperitoneal injections of 100 mg/kg NVP-AUY-922 or
vehicle. Ex vivo, tumour c-MET immunohistochemistry was
correlated with the imaging results.
Results In vitro, membrane c-MET was upregulated in
HCC827ErlRes tumours by 213 ± 44% in relation to the level
in HCC827 tumours, while c-MET was downregulated by 69
± 9% in HCC827 tumours following treatment with
NVPAUY-922. In vivo, 89Zr-onartuzumab uptake was 26% higher
(P < 0.05) in erlotinib-resistant HCC827ErlRes than in
HCC827 xenografts, while HCC827 tumour uptake was
33% lower (P < 0.001) following NVP-AUY-922 treatment.
Conclusion The results show that 89Zr-onartuzumab PET
effectively discriminates relevant changes in c-MET levels and
could potentially be used clinically to monitor c-MET status.
c-MET is a receptor tyrosine kinase with roles in
embryogenesis, and tissue repair and regeneration in homeostasis.
Dysregulation of c-MET is often found in solid cancers as a
consequence of mutation, protein overexpression, gene
amplification and auto/paracrine upregulation of its ligand
hepatocyte growth factor (HGF). Aberrant activation of c-MET/HGF
signalling leads to increased tumour proliferation,
invasiveness and angiogenesis, resulting in poor prognosis in various
human cancers, including non-small-cell lung cancer
(NSCLC) and gastric cancer . NSCLC patients harbouring
mutations activating epidermal growth factor receptor
(EGFR) can be treated with the EGFR tyrosine kinase
inhibitors (TKIs) erlotinib and gefitinib. Resistance of
NSCLC to EGFR TKIs invariably occurs in these patients
due to secondary mutations or activation of bypass signalling
pathways, such as c-MET/HGF [2, 3]. Both c-MET and HGF
have been implicated in resistance to EGFR-targeted therapies
due to crosstalk via shared downstream signalling
intermediates [2, 4]. Concurrent transient exposure to HGF can result in
lasting resistance to EGFR targeting TKIs by providing
positive selection pressure for c-MET-upregulated resistant
subclones [5, 6]. c-MET-mediated resistance in EGFR
TKIresistant NSCLC was detected in around 5–15% of patients
using quantitative polymerase chain reaction and fluorescence
in situ hybridization [2, 7].
Several c-MET-directed treatment strategies have been
reported. NVP-AUY-922, a non-geldanamycin heat shock
protein 90 (HSP90) inhibitor, can potently downregulate EGFR
and c-MET, and can overcome c-MET-mediated resistance in
an EGFR TKI-resistant clone of NSCLC cell line HCC827
. NVP-AUY-922 is currently in clinical trials in advanced
E G F R m u t a n t N S C L C r e f r a c t o r y t o E G F R T K I s
(NCT01646125, NCT01124864), or with an EGFR exon 20
mutation (NCT01854034). The combination of
NVP-AUY922 and erlotinib in a phase I/II study of erlotinib-treated
patients with progressive NSCLC has shown a 16% partial
tumour response rate . Another form of c-MET-directed
therapy involves antibodies. Onartuzumab, is a 99-kDa
onearmed antibody, reactive to human but not murine c-MET.
Onartuzumab inhibits HGF binding, blocks c-MET receptor
phosphorylation and downstream signalling and has shown
antitumour efficacy in preclinical models .
In a randomized phase II trial in patients with advanced
NSCLC, the addition of onartuzumab to erlotinib resulted in
an improvement in progression-free and overall survival in
patients positive for c-MET on immunohistochemistry (IHC)
. However, the METLung randomized phase III clinical
trial in which erlotinib was combined with onartuzumab or
placebo in patients with NSCLC and positive for c-MET on
IHC was terminated early because of lack of effectiveness
. MET amplification and overexpression, on the other
hand, are associated with increased responses to c-MET
inhibitors . Recently, splice alterations in exon 14 of the MET
gene (METex14), that are found in about 3% of human lung
cancers, have been found to be associated with in vitro and
clinical sensitivity to the c-MET TKI capmatinib and the
ALK/ROS1/MET TKI crizotinib . METex14 leads to
impaired c-MET downregulation and degradation, resulting in
cMET protein overexpression .
Selection of patients with c-MET amplification or
overexpression could therefore potentially be beneficial for
therapy decisions, due to their sensitivity to
c-METdirected agents. IHC is generally performed on archived
tissues and therefore reflects c-MET status of the tissues
at the time of retrieval. c-MET status, however, can vary
over time and among lesions, indicating a need for
biomarkers able to capture this plasticity for the body as a
whole [13, 16]. PET may provide a noninvasive method
for assessing whole-body c-MET status to capture c-MET
dynamics after the emergence of c-MET-mediated EGFR
TKI resistance, or as response to HSP90 inhibition.
For ease of clinical translation, we selected the
therapeutic antibody-based PET tracer 89Zr-onartuzumab,
which has been reported to effectively discriminate
cMET expression in human gastric cancer xenografts
. Several other antibody and antibody fragment tracers
have been reported to be of value for preclinical c-MET
PET imaging . These studies used an immunogenic
full-length murine antibody DN30  or antibody
fragments and other protein scaffolds such as the 89Zr-labelled
H2 cys-diabody, H2 minibody , and anticalin
89ZrPRS110 . In contrast to 89Zr-onartuzumab, these
tracers are not readily translatable to the clinic, as none
of these tracer protein backbones has yet been
administered to patients, and would therefore require extensive
safety testing. Furthermore, administration of tracers
based on the HGF ligand such as 64Cu-HGF, as well as
the bivalent antibody DN30 [19, 22], might have the
unwanted result of stimulating tumour growth by activating
We therefore aimed to validate the ability of
89Zronartuzumab PET to assess c-MET upregulation-mediated
erlotinib resistance, as well as c-MET downregulation after
HSP90 inhibitor NVP-AUY-922 treatment in human
Materials and methods
Cell lines and chemicals
The human NSCLC cell line HCC827 was obtained from the
American Type Culture Collection. Cells were authenticated
in April 2015 by STR profiling using BaseClear and
quarantined until screening showed that they were negative
for microbial and mycoplasma contamination. Cells were
subcultured twice weekly using Roswell Park Memorial
Institute-1640 (RPMI-1640) medium supplemented with
10% fetal calf serum (Bodinco BV) and incubated at 37 °C
in a fully humidified atmosphere containing 5% CO2.
HCC827ErlRes, a stable erlotinib-resistant subclone, was
generated by culturing the parental cell line HCC827 in the
presence of 1 μM erlotinib and 50 ng/mL recombinant human
HGF (PeproTech) for 2 weeks, followed by 2 weeks in
1 μM erlotinib, a method analogous to that described by
Turke et al. . NVP-AUY-922 (Luminespib; LC
Laboratories) and erlotinib (LC Laboratories) were dissolved
in dimethyl sulfoxide, and aliquots stored at −80 °C.
In vitro cell analyses
Surface expression of EGFR and c-MET was assessed using a
BD Accuri™ C6 flow cytometer (BD Biosciences).
Cetuximab (5 mg/mL; Merck) and onartuzumab (60 mg/mL;
Genentech) served as primary antibodies for EGFR and
cMET, respectively, and mouse anti-human Fc-specific
FITCconjugated secondary antibody (clone HP-6017;
SigmaAldrich) was used for readout of both EGFR and c-MET
expression, with 10,000 events assessed per sample. The
sensitivity of HCC827 and HCC827ErlRes cell lines to erlotinib
and NVP-AUY-922 after 4 days of treatment was determined
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay .
Onartuzumab was conjugated at five molar ratios (1:1, 1:2,
1:3, 1:5 and 1:10) of antibody to
tetrafluorophenol-Nsuccinyldesferal-Fe3+ (Df; ABX GmbH), in triplicate for each
ratio, as described previously . Df-onartuzumab
conjugates were checked for aggregation and fragmentation by
size-exclusion high-performance liquid chromatography
(SE-HPLC) using a Waters system equipped with a dual
wavelength absorbance detector, in-line radioactivity detector
and a TSK-GEL G3000SWXL column (JSB).
Phosphatebuffered saline (PBS; 140 mmol/L NaCl, 9 mmol/L
Na2HPO4, 1.3 mmol/L NaH2PO4; pH 7.4) was used as the
mobile phase. The ratio of conjugated
tetrafluorphenol-Nsuccinyldesferal-Fe3+ to antibody was determined by the
antibody-bound versus unbound 430 nm signal of Fe3+ on
89Zr labelling was performed as described previously 
using clinical grade 89Zr-oxalate (Perkin Elmer). Maximum
attainable specific activity was determined by radiolabelling
Df-onartuzumab conjugates with specific activities between
50 and 1,000 MBq 89Zr per milligram of conjugate. The
radiochemical purity (RCP) of 89Zr-labelling was assessed by
the trichloroacetic acid precipitation test . The stability of
89Zr-onartuzumab was tested by incubation for 7 days under
the following conditions: 0.9% NaCl at 4 °C, human serum at
37 °C and 0.5 M HEPES buffer, pH 7.2, at 37 °C.
The immunoreactivity of Df-onartuzumab conjugates was
assessed by a competition assay as described previously. In
brief, NuncBreakApart 96-well plates were coated overnight
at 4 °C with 100 μL 100 ng/mL c-MET extracellular domain
(Genentech) in 0.1 M Na2CO3 buffer, pH 9.6.
89ZrOnartuzumab was diluted to 5,000 ng/mL in assay diluent
which consisted of PBS, 0.5% bovine serum albumin fraction
V (Bio-Connect) and 0.05% Tween 20 (Sigma-Aldrich).
Plates were washed with washing buffer (PBS with 0.05%
Tween 20) and blocked by incubation for 1 h at room
temperature with 200 μL assay diluent, and washed again.
Eight dilutions of unmodified onartuzumab at molar excesses
between 0.004 and 156.25-fold were premixed 1:1 with
diluted 89Zr-onartuzumab solution, and 100 μL aliquots of the
mixtures were incubated in wells for 1 h at room temperature.
After washing, the wells were broken apart and counted using
a calibrated well-type 1282 Compugamma system (LKB
Wallac). Counts were plotted against concentrations of
competing unmodified onartuzumab and the half-maximum
inhibitory concentration (IC50) was calculated using GraphPad 5.0
(GraphPad Software, Inc.). The IC50 was divided by the final
tracer concentration (2,500 ng/mL) to yield the
111In-OA-NBC and 89Zr-OA-NBC control tracer
To determine nonspecific tracer distribution in organs, as well
nonspecific tumour uptake, a one-armed isotype control
antibody OA-NBC (Genentech) was coinjected and used as
control tracer in all experiments. For 111In labelling, OA-NBC
was conjugated with p-SCN-Bn-DTPA (Macrocyclics) as
described previously . Radiolabelling was performed with
111In-chloride (Mallinckrodt). The RCP of 111In-OA-NBC
labelling was checked by silica gel instant thin-layer
chromatography (ITLC) using 0.1 M citrate buffer, pH 6.0, as eluent.
Furthermore, OA-NBC was incubated with a 1:5 molar excess
of OA-NBC to Df, in a procedure similar to that described for
Df-onartuzumab, to yield Df-OA-NBC conjugate for
Male nude mice (BALB/cOlaHsd-Foxn1nu; Envigo) were
inoculated subcutaneously with xenograft tumours. Mice
bearing only HCC827 xenograft tumours were injected
subcutaneously with 5 × 106 HCC827 cells into the right flank.
To determine the optimal tracer protein dose and imaging
time-point, three groups each of four to six mice inoculated
with HCC827 xenografts received 10, 25 or 100 μg
89Zronartuzumab (effective injected protein doses 9.7 ± 0.2, 24.0
± 0.3 and 96.7 ± 0.9 μg at 4.24 ± 0.20, 4.57 ± 0.15 and 4.76 ±
0.13 MBq, respectively) coinjected with an equal amount of
111In-OA-NBC (1 MBq) isotype control via a penile vein.
Tumour sizes were 183 ± 63, 234 ± 46 and 180 ± 92 mm3 in
the groups treated with 10, 25 and 100 μg, respectively. All
tracer injections contained 10 μg radiolabelled
89Zronartuzumab (specific activity about 400–500 MBq/mg) and
10 μg 111In-OA-NBC (specific activity about 100 MBq/mg),
with cold onartuzumab and OA-NBC added to reach the total
stated protein doses. Another group of three mice received
10 μg 89Zr-OA-NBC (effective protein doses 9.7 ± 0.3 μg at
4.47 ± 0.13 MBq) coinjected with 10 μg 111In-OA-NBC
(1 MBq) to validate the use of 111In-OA-NBC as a proxy for
the 89Zr-labelled control molecule. MicroPET scans were
performed 1, 3 and 6 days after injection, followed by ex vivo
biodistribution analysis after the final scan.
c-MET expression on HCC827ErlRes cells in comparison
with that on HCC827 cells was evaluated by inoculating a
group of ten mice with 5 × 106 HCC827 cells into the right
flank and 5 × 106 HCC827ErlRes cells into the opposite flank.
The sizes of the HCC827 and HCC827ErlRes tumours were
457 ± 182 and 240 ± 74 mm3 (P < 0.01), respectively, at the
time of biodistribution analysis (day 6). Mice received 10 μg
89Zr-onartuzumab (effective protein dose injected 9.7 ± 0.2 μg
at 4.42 ± 0.09 MBq) coinjected with 10 μg 111In-OA-NBC
(1 MBq) via a penile vein. MicroPET scans were performed
6 days after injection, followed by ex vivo biodistribution
The effects of HSP90 inhibition on c-MET expression were
evaluated in vivo using HCC827 xenograft-bearing mice.
Mice bearing tumours of sizes 159 ± 65 and 208 ± 112 mm3
received 10 μg 89Zr-onartuzumab (effective protein dose
injected 9.7 ± 0.1 and 9.8 ± 0.1 μg at 4.68 ± 0.15 and 4.44 ±
0.22 MBq for NVP-AUY-922 and vehicle, respectively)
coinjected with 10 μg 111In-OA-NBC (1 MBq) via a penile
vein on day 0. Scans were performed on day 6, after which the
mice were treated by intraperitoneal injection with either
100 mg/kg NVP-AUY-922 in 5% glucose (seven mice) or
5% glucose vehicle (six mice) on days 6, 10, 13, 16 and 19.
Mice received a second identical tracer injection on day 13
(effective protein dose injected 9.7 ± 0.1 and 9.6 ± 0.2 μg at
4.45 ± 0.36 and 4.41 ± 0.37 MBq for NVP-AUY-922 and
vehicle, respectively), followed by microPET scans on day 19
and ex vivo biodistribution analysis. Tumours measured 205
± 127 mm3in NVP-AUY-992-treated mice and 313 ± 198 mm3
in vehicle-treated mice at the time they were euthanised.
All microPET scans were performed using a Focus 220
rodent scanner (CTI Siemens). MicroPET scans were
reconstructed and in vivo quantification was performed using
AMIDE v. 1.0.4 . Regions of interest (ROI) were drawn
on the tumour based on the ex vivo weight assuming 1 g/cm3
tissue density or measured tumour volume for longitudinal
experiments. Scan data are presented as mean standardized
uptake values (SUVmean), which were calculated from the
mean activity in the ROI divided by the injected dose
(corrected for decay) per gram body weight, as described
previously . For biodistribution studies, organs and injected
tracer standards were counted using the calibrated well-type
LKB 1282 Compugamma system and weighed. 111In and 89Zr
were counted in a single measurement at 311–500 keV and
758–1,144 keV, respectively, with crosstalk in the 111In
channel corrected using a reference 89Zr dilution series. After
decay correction, ex vivo tissue activities were expressed as the
percentages of injected dose per gram tissue (%ID/g).
Xenograft tumours were formalin-fixed and
paraffinembedded for IHC analysis. All animal experiments were
approved by the Institutional Animal Care and Use
Committee of the University of Groningen.
Formalin fixed, paraffin-embedded tissue slices of thickness
4 μm were stained for c-MET using monoclonal rabbit
antihuman c-MET antibody diluted 1:400 (ab51067; Abcam)
. Haematoxylin and eosin (H&E) staining was performed
regularly to assess tissue viability and morphology. Digital
scans of slides were acquired using a NanoZoomer 2.0-HT
multislide scanner (Hamamatsu) and analysed with
NanoZoomer Digital Pathology viewer software.
Data are presented as means ± SD. Statistical analyses were
performed with GraphPad Prism 5.0 using the two-sided
Mann–Whitney test for nonparametric data and the
twosided paired Student’s t test for paired data. P values ≤0.05
were considered significant.
Effects of erlotinib resistance and NVP-AUY-922
treatment on c-MET expression
An erlotinib-resistant clone, HCC827ErlRes, was generated
from the parental cell line HCC827 by culturing cells for
2 weeks with 50 ng/mL HGF and 1 μM erlotinib, followed
by 2 weeks culture in the presence of 1 μM erlotinib. Surface
expression of c-MET on HCC827ErlRes cells as measured by
flow cytometry was upregulated to 213 ± 44%, while EGFR
surface levels were downregulated to 35 ± 17% of levels in the
parental HCC827 cells (Fig. 1a). HCC827ErlRes cells were
able to fully proliferate in the presence of up to 1,000 nM
erlotinib as measured by the MTT assay, while parental
HCC827 cells remained highly sensitive to erlotinib with an
IC50 of 12 nM (Fig. 1b). NVP-AUY-922 treatment reduced
surface expression of EGFR and c-MET (Fig. 1c).
NVPAUY-922 treatment was equally effective in reducing the
viability of both HCC827 and HCC827ErlRes cells (Fig. 1d).
89Zr-onartuzumab tracer development
Conjugation of Df to onartuzumab was approximately 60%
efficient for all molar reaction ratios tested (Supplementary
Fig. 1a). Df-onartuzumab conjugates were able to consistently
bind ≥500 MBq 89Zr per milligram of Df-onartuzumab with
RCP ≥95% at ratios above 1:2 onartuzumab bound to Df
(Supplementary Fig. 1b). The competition assay revealed a
trend for lower immunoreactivity at higher conjugation ratios,
Fig. 1 a In vitro flow cytometric analysis of EGFR and c-MET
membrane expression in HCC827ErlRes cells normalized to expression
in parental cell line HCC827. b In vitro MTT proliferation assay in
HCC827 and HCC827ErlRes cells with exposure to increasing
concentrations of erlotinib for 4 days. c In vitro flow cytometric
analysis of EGFR and c-MET membrane expression in HCC827 and
HCC827ErlRes cells after 24 h treatment with 25, 50 and 100 nM
NVP-AUY-922 normalized to untreated controls. d In vitro MTT
proliferation assay in HCC827 and HCC827ErlRes cells with exposure
to increasing concentrations of NVP-AUY-922 for 4 days
signifying a need for balancing the required specific activity
with the retained affinity of Df-onartuzumab conjugates
(Supplementary Fig. 1c). Aggregation and fragmentation of
Df-onartuzumab conjugates were not observed. A conjugation
reaction ratio of 1:5, yielding 3.11 ± 0.33 Df bound to
onartuzumab, was chosen as the optimal ratio for animal
studies. 89Zr-Onartuzumab was stable in vitro, with a maximum
observed decrease in RCP from 99.0 ± 0.2% to 91.0 ± 6.6% in
human serum after 7 days at 37 °C (Supplementary Fig. 1d).
All 89Zr-onartuzumab tracer batches had a RCP of ≥95% by
trichloroacetic acid precipitation, while 111In-OA-NBC
batches had a RCP of ≥90% by ITLC.
89Zr-Onartuzumab protein dose escalation
89Zr-Onartuzumab tumour uptake increased over time for all
tracer protein doses tested, with the highest tumour and least
background organ uptake observed on day 6 after injection
(Fig. 2a). 89Zr-Onartuzumab tumour uptake was higher than
that of the coinjected 111In-OA-NBC control tracer in all tracer
protein dose groups (Fig. 2b, Supplementary Fig. 2a, b). The
10 and 25 μg 89Zr-onartuzumab dose groups showed similar
tumour uptakes of 31.6 ± 8.7 and 29.8 ± 12.1%ID/g, while
uptake in the 100 μg 89Zr-onartuzumab dose group was lower
but not significantly (P = 0.17 vs. the 10 μg group) at 23.5 ±
9.4%ID/g. SUVmean values in tumours of the 10, 25 and
100 μg 89Zr-onartuzumab dose groups were 2.7 ± 0.9, 2.9 ±
0.7 and 2.2 ± 0.8, respectively (Fig. 2b, c, Supplementary
Fig. 2a, b). The 89Zr-OA-NBC control showed no
accumulation in HCC827 tumours over time (Fig. 2a, c, Supplementary
Fig. 2a, b), while ex vivo tumour uptake was similar to that of
111In-OA-NBC (Fig. 2b, Supplementary Fig. 2a, b), showing
the suitability of 111In-OA-NBC as a proxy for 89Zr-OA-NBC.
Based on these results, a 89Zr-onartuzumab protein dose of
10 μg was used in the next mouse cohorts.
PET scans showed 24% higher 89Zr-onartuzumab uptake in
HCC827ErlRes tumours (SUVmean 3.3 ± 0.5) than in
HCC827 tumours (SUVmean 2.7 ± 0.3; Fig. 3a, b; P < 0.01).
Biodistribution analysis revealed that 89Zr-onartuzumab
uptake was 26% higher in HCC827ErlRes tumours (38.1 ±
8.4%ID/g) than in HCC827 tumours (30.2 ± 3.5%ID/g;
Fig. 3c, Supplementary Fig. 3, 6; P < 0.05). Similar
comparisons of tumour-to-muscle and tumour-to-blood ratios for the
two groups showed similar trends, but the differences did not
reach significance. IHC revealed similar levels of necrosis, as
well as higher c-MET expression in HCC827ErlRes than
HCC827 tumours, in accordance with the 89Zr-onartuzumab
PET and biodistribution data (Fig. 3d, Supplementary Fig. 5a).
Fig. 2 a Representative microPET scans of mice bearing HCC827
xenografts 24, 72 and 144 h after injection of 10, 25 and 100 μg
89Zronartuzumab (six, five and four mice, respectively) and 10 μg
89Zr-OANBC (three mice). b Ex vivo tumour uptake in HCC827 tumours 6 days
after injection of 10, 25 and 100 μg 89Zr-onartuzumab and 10 μg
89Zr-OA-NBC compared with the 111In-OA-NBC control. c MicroPET
quantification of uptake in HCC827 tumours 24, 72 and 144 h after
injection of 10, 25 and 100 μg 89Zr-onartuzumab and 10 μg
Fig. 3 a Representative microPET scans in a representative mouse 144 h
after injection of HCC827 cells (P, right flank) and HCC827ErlRes cells
(ER, left flank). b MicroPET quantification of 89Zr-onartuzumab uptake
in HCC827 and HCC827ErlRes tumours 144 h after injection of
HCC827 and HCC827ErlRes cells. c Ex vivo uptake of
89Zro n a r t u z u m a b a n d 111 I n - O A - N B C c o n t r o l i n H C C 8 2 7 a n d
HCC827ErlRes tumours. d Ex vivo tissue analysis. c-MET and
haematoxylin and eosin immunohistochemical staining in HCC827 and
The effects of NVP-AUY-922 treatment on c-MET expression
were evaluated in HCC827 xenograft-bearing mice. In mice
receiving NVP-AUY-922, the tumour SUVmean for
89Zronartuzumab was 33 ± 10% lower after treatment than in the
baseline scans (P < 0.001), while uptake values in
vehicletreated mice before and during treatment were similar
(Fig. 4a, b). Biodistribution studies confirmed the PET results
revealing 27% lower 89Zr-onartuzumab tumour uptake in
NVP-AUY-922-treated mice than in vehicle-treated mice
(Fig. 4c, Supplementary Figs. 4a, 4b, 6; P < 0.05). Similar
comparisons of the tumour-to-muscle and tumour-to-blood
ratios for the two groups showed similar trends, but the
differences did not reach significance. IHC showed a marked
decrease in c-MET expression in the tumours, with similar
levels of necrosis, in NVP-AUY-922-treated and in
vehicletreated animals (Fig. 4d, Supplementary Fig. 5b).
This study illustrates the feasibility of in vivo imaging of
cMET dynamics by detecting upregulation after
c-METmediated erlotinib resistance, as well as downregulation of
c-MET following HSP90-directed therapy using
89Zronartuzumab PET in human NSCLC xenograft-bearing mice.
Monitoring c-MET in vivo with 89Zr-onartuzumab PET might
be an attractive method for detecting c-MET-mediated
emergence of resistance to EGFR TKIs, as well as for assessing
downregulation of c-MET in response to HSP90 inhibition.
Furthermore, it could be beneficial for patient selection for
cMET-directed therapy, as MET amplification and
overexpression are associated with increased response to c-MET
Because of the different Df-suc-N-onartuzumab linker
chemistry used for 89Zr radiolabelling in the present study
and for 89Zr-Df-Bz-SCN-onartuzumab in the study by
Jagoda et al. , we performed a tracer protein dose
escalation study to optimize the 89Zr-onartuzumab protein dose and
Fig. 4 a Representative microPET scans in mice bearing HCC827
xenografts 144 h after injection, before treatment (day 6) and during
treatment (day 19) with vehicle (six mice) or 100 mg/kg
NVP-AUY922 (seven mice). b MicroPET quantification of uptake in HCC827
tumours before treatment (day 6) and during treatment (day 19) with
vehicle or 100 mg/kg NVP-AUY-922. c Ex vivo HCC827 tumour
uptake (day 19) in vehicle-treated and NVP-AUY-922-treated mice (as
%ID/g 89Zr-onartuzumab and 111In-OA-NBC control). d c-MET and
haematoxylin and eosin immunohistochemical staining of tumours from
vehicle-treated and NVP-AUY-922-treated mice
imaging time-point. HCC827 tumours in the present study
showed high contrast and c-MET-specific 89Zr-onartuzumab
uptake at a tracer protein dose of 10 μg. Higher
89Zronartuzumab protein doses showed a trend for lower tumour
uptake. This blocking effect was also shown by Jagoda et al.
using 1 mg cold onartuzumab, and is most likely caused by its
nonreactivity with murine c-MET [10, 17]. 89Zr-Onartuzumab
is remarkable in that respect, in that specific binding can be
proven in vivo in a conventional blocking study. In contrast,
cross-reactive antibodies might need a higher protein dose for
optimal tumour contrast . Similar protein doses of
89ZrOA-NBC and 111In-OA-NBC one-armed isotype controls for
89Zr-onartuzumab did not accumulate in tumour tissues,
confirming the c-MET specificity of the 89Zr-onartuzumab
signal. 89Zr-Df-p-SCN-onartuzumab uptake in MKN-45
gastric cancer xenografts 5 days after injection was lower at
22.5%ID/g than observed for 89Zr-Df-suc-N-onartuzumab in
HCC827 xenografts . A direct comparison, however, is
difficult due to differences in tumour models, desferal linker
chemistries, tracer protein doses and day of biodistribution
Upregulation of c-MET in EGFR TKI gefitinib-resistant
HCC827-GR6 xenografts was visualized with a 89Zr-labelled
H2 cys-diabody and H2 minibody, with twofold higher uptake
o b s e r v e d i n r e s i s t a n t t u m o u r s [ 2 0 ]. The r eported
biodistribution data for the resistant tumours, however,
excluded cystic areas, which affected approximately half of the
resistant c-MET-upregulated HCC827-GR6 tumours.
Exclusion of these cystic areas might have artificially
increased the apparent uptake of these tracers. Furthermore, a
highly variable absolute tumour uptake was observed,
differing by up to twofold between experiments, correlating with a
similar variation in the remaining blood pool activity . We
observed robust tumour uptake of 89Zr-onartuzumab. As the
affinity of the H2 cys-diabody and H2 minibody for c-MET
are comparable to that of onartuzumab, this is probably due to
the high molecular weight of 89Zr-onartuzumab that results in
slower blood clearance and prolonged tumour exposure [10,
20]. 89Zr-Onartuzumab tumour-to-muscle and
tumour-toblood ratios showed similar trends, but the differences
between HCC827 and HCC827Erlres tumours did not reach
significance. However, we included the 111In-OA-NBC paired
control molecule in each experimental animal as a valid
control to determine specific tumour uptake , as well as
ex vivo analysis for c-MET expression and necrosis. This
strengthens the conclusion that the extra uptake in
c-METupregulated HCC827ErlRes xenografts is indeed mediated
by c-MET expression, and is not caused by possible variations
in tumour size, blood pool tracer availability and permeability
Pharmacokinetic readout of c-MET in response to c-MET
TKI PHA665752 therapy has shown reduced total MET
protein in human gastric cancer MKN-45 xenografts due to more
necrosis which coincides with lower uptake of c-MET-specific
peptide 99MTc-AH-113018 . The AH113804 peptide has
also been labelled with 18F for PET, and is able to detect
recurrence of c-MET-expressing basal-like breast cancer
xenografts after surgical resection . A fluorescent labelled
version of the same peptide has been used as a ‘red flag’
technique to detect polyps using a fluorescence endoscope
technique in patients at risk of developing colon cancer .
We and others have shown potent downregulation of both
EGFR and c-MET in human NSCLC cell lines by exposure to
HSP90 inhibitors NVP-AUY-922 and 17-DMAG [8, 35].
cMET downregulation by treatment with NVP-AUY-922 has
also been shown to overcome c-MET-mediated gefitinib and
erlotinib acquired resistance in HCC827 sub clones . We
have visualized this downregulation of c-MET by treatment
with NVP-AUY-922 in vivo using 89Zr-onartuzumab PET,
with sequential scans showing 33% lower uptake after
treatment. This correlated with downregulation of c-MET protein
on ex vivo IHC, while tumour necrosis levels were similar in
NVP-AUY-922-treated and vehicle-treated animals.
A limiting factor in the present study may have been the
relatively small effect sizes observed for upregulation and
downregulation of tracer tumour accumulation. Preclinical
studies using 89Zr-labelled antibody tracers have shown that
HSP90 inhibition and everolimus treatment downregulate
other key oncogenic proteins, such as HER2, vascular endothelial
growth factor A (VEGF-A), and insulin-like growth factor 1
receptor (IGF1R). The effect sizes of antibody tracer uptake
reduction after target protein downregulation through HSP90
inhibition and everolimus treatment are comparable to the
reduction in uptake of 89Zr-onartuzumab after c-MET
downregulation found in the present study [36–39], and the results
of some of these preclinical studies have been translated to
successful treatments in the clinic [40, 41]. Furthermore,
insight into whole-body c-MET target distribution via
noninvasive 89Zr-onartuzumab scans could potentially enlarge the
patient population which might benefit from
c-MET/HGFtargeted drugs .
Based on these promising preclinical results, we conclude
that 89Zr-onartuzumab c-MET PET provides a robust
noninvasive and powerful tool that is easily translatable to the clinic
for visualizing c-MET dynamics as a possible biomarker for
cMET-mediated resistance to erlotinib and for treatment
response to HSP90 inhibition.
Acknowledgments The authors thank Dr. Simon-Peter Williams of
Genentech Inc. for his assistance.
Compliance with ethical standards
Conflict of interests None.
Grant support This work was funded by European Research Council
(ERC) advanced grant OnQview.
Ethical approval All applicable international, national, and/or
institutional guidelines for the care and use of animals were followed. This
article does not describe any studies with human participants performed
by any of the authors.
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