5th EuropEan MolEcular IMagIng MEEtIng - ESMI

5th EuropEan MolEcular IMagIng MEEtIng – EMIM2010
Pre-clinical screening of anti - HER2 nanobodies for molecular imaging of breast cancer
Vaneycken I. (1) , Devoogdt N. (1) , Peleman C. (1) , Xavier C. (1) , Lahoutte T. (1) , Caveliers V. (1) .
Vrije Universiteit Brussel, Belgium
ilse.vaneycken@gmail.com
Introduction: Nanobodies are small antigen-binding
fragments of camelid heavy-chain antibodies. Besides
therapeutic agents, they are also effective as a
diagnostic tool for targeted imaging when labeled
with a suitable radionuclide[1]. We produced Nanobodies
for molecular imaging of Human Epidermal
Growth Factor Receptor 2 (HER2) expression in
breast cancer patients[2] for a phase I clinical trial.
After production, a number of candidate binders
are obtained. we designed a standardized selection
procedure based on production yield, in vitro and
in vivo criteria to identify the lead anti-HER2 Nanobody
with the highest potential as a tracer for clinical
translation.
Methods: A camel was immunized with HER2 recombinant
protein and serum IgG2 and IgG3 subclasses
with heavy-chain-only antibodies were
separated. Eleven different anti-HER2 Nanobodies
were produced in E. coli, purified and labeled with
99mTc(I)-tricarbonyl. In vitro saturation binding
studies were performed on recombinant HER2 and
HER2 positive SKOV3 cells with the native and radiolabeled
anti-HER2 Nanobodies. Competition
studies allowed assessing whether the anti-HER2
99mTc-Nanobodies competed with the therapeutic
anti-HER2 antibodies Trastuzumab and Pertuzumab.
In vivo, the biodistribution and tumor targeting
potential of all 99mTc-Nanobodies was evaluated
using pinhole SPECT/micro-CT in nude mice bearing
HER2 positive SKOV3 xenografts. The Nanobody
showing the most favourable in vivo characteristics
was additionaly evaluated in nude mice
bearing HER2 positive LS174T and HER2 negative
MDAMB4357d xenografts.
Results: Nanobodies were labeled with 99mTc and
purified to a final radiochemical purity of ≥99%.
Saturation binding studies showed that 99mTc labeling
was not associated with a significant reduction of
immunoreactivity. The Nanobodies appeared to be
not or only weakly competitive with the commercial
therapeutic antibodies. In vivo biodistribution demonstrated
that tumor accumulation varied between
0,78 and 4,44 percent injected activity per gram
(%IA/g). Of the eleven 99mTc-Nanobodies tested in
SKOV3 xenografts, three presented a tumor uptake
above 4 %IA/g. One was selected and also showed
high tumor uptake (3,76±0,82 %IA/g) in LS174T xenografts,
but low uptake in MDAMB435d xenografts
(0,71±0,07 %IA/g). In addition, all 99mTc-Nanobodies
displayed high renal uptake but low non-specific
accumulation in liver, muscle and blood, resulting
in high tumor-to-background ratios.
Conclusions: Using a standardized selection procedure
for identificiation of high affinity Nanobodies
for molecular imaging of cancer, we identified one
Nanobody as the lead compound for a phase I clinical
trial. This Nanobody meets with all criteria that
characterize a good diagnostic tracer.
Acknowledgement: The research at ICMI is funded
by the Interuniversity Attraction Poles Program–
Belgian State–Belgian Science Policy. Tony Lahoutte
is a Senior Clinical Investigator of the Research
Foundation–Flanders (Belgium) (FWO).
References:
1. Gainkam LO, Huang L, Caveliers V, et al. Comparison of
the biodistribution and tumor targeting of two 99mTclabeled
anti-EGFR nanobodies in mice, using pinhole
SPECT/micro-CT. J Nucl Med. 2008;49(5):788-795.
2. Hicks,D.G. et al (2008). HER2+ breast cancer: review of
biologic relevance and optimal use of diagnostic tools.
American Journal of Clinical Pathology, 129, 263-273.
EuropEan SocIEty for MolEcular IMagIng – ESMI
YIA applicant
day1
Parallel Session 1: CANCER I - together with the ESR