JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Imaging and Biodistribution of Her2/Neu Expression in
Non-Small Cell Lung Cancer Xenografts with
64 Cu-labeled Trastuzumab PET
P. Paudyal a) , B. Paudyal a) , H. Hanaoka a) , N. Oriuchi a) , Y. Iida a) , H. Yoshioka a) ,
H. Tominaga a) , Sa. Watanabe b) , Sh. Watanabe b) , N. S. Ishioka b) and K. Endo a)
a) Graduate School of Medicine, Gunma University,
b) Radiation-Applied Biology Division, QuBS, JAEA
Lung cancer is one of the leading causes of death with a
5-year survival rate of less than 10%. Activation of human
epidermal growth factor receptor 2 (Her2/neu) genes is
encountered in subpopulations of non-small cell lung
carcinomas (NSCLC). NSCLC overexpress the Her2/neu
gene in approximately 59% of cases. Trastuzumab, a
humanized monoclonal antibody, interferes with Her2
signaling and is approved for the treatment of Her2/neu
overexpressing breast cancer. However, its therapeutic use
in Her2/neu overexpressing NSCLC remains obscure 1) .
64
Cu with half-life 12.7 h labeled monoclonal antibody
has been garnering interest in the field of targeted imaging
due to its emission of both β + (17.4%) and β - (41%) 2) . The
present study aimed to determine the role of 64 Cu-labeled
trastuzumab positron emission tomography (PET) for
non-invasive imaging of Her2/neu expression in NSCLC.
Methods: Trastuzumab was conjugated with the
bifunctional chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4,
7, 10-tetraacetic acid (DOTA) and radiolabeled with 64 Cu.
The molecular specificity of DOTA-trastuzumab was
determined in NSCLC cell lines with Her2/neu
overexpression (NCI-H2170) and negative expression
(NCI-H520). 64 CuCl2 was provided in a dry state and was
dissolved in sodium acetate buffer (0.25 M, pH 6.0).
Thirty μg of DOTA-trastuzumab was added and the mixture
was incubated at 40 ºC for 1.5 h. Then, 10 mM EDTA was
added and the above solution was incubated for 15 min at
45 ºC. The labeling yield was checked with thin layer
chromatography (TLC). The final purification was done
using a Bio-Spin 6 Tris column. Imaging of Her2/neu
expression was performed in NCI-H2170 tumor-bearing
mice with
64 Cu-DOTA-trastuzumab PET and
64
Cu-DOTA-IgG. Size exclusion HPLC (SE-HPLC) was
used to evaluate the in vitro and in vivo stability of
64
Cu-DOTA-trastuzumab. For in vitro stability,
64
Cu-DOTA-trastuzumab was mixed with murine serum and
the solution was incubated at 37 C and the aliquots were
analyzed at 1 h, 24 h and 48 h. For in vivo analysis, blood
was drawn from the mice injected with
64
Cu-DOTA-trastuzumab at 1 h and 48 h and the sample was
analyzed by SE-HPLC.
Results: The labeling efficiency of
64 Cu-DOTA-
trastuzumab was 92% without purification and 99% after
purification, which should allow the specific uptake of the
conjugate. The number of DOTA chelator molecule
attached to the trastuzumab was 5-6 per mole of trastuzumab.
Incubation of 64 Cu-DOTA-trastuzumab in murine and in
vivo revealed that the conjugate existed only in the intact
form (retention time : 19-20 min) and no transchelation to
protein was seen until 48 h which confirmed the stability of
the conjugate as shown in the Fig. 1. The retention time of
the sample, serum and blood was 19-20 min. In vitro
studies revealed specific binding of DOTA-trastuzumab in
JAEA-Review 2010-065
the Her2/neu positive NCI-H2170 cells, while no binding
was seen in the Her2/neu negative NCI-H520 cell line.
Biodistribution and PET studies revealed a significantly
high accumulation of 64 Cu-DOTA-trastuzumab in the
Her2/neu overexpressing NCI-H2170 tumor at 24 h and 48 h
post-injection (21.4 ± 1.4% and 23.2 ± 5.1% injection
dose/gram (% ID/g), respectively) as shown in the Fig. 2.
PET imaging of Her2/neu negative NCI-H520 tumors
showed much less uptake of 64 Cu-DOTA-trastuzumab (4.0%
ID/g). The NCI-H2170 tumor uptake of 64 Cu-DOTA-
trastuzumab was significantly higher than that of
64 Cu-DOTA-IgG (p