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Positron-Emission Tomography (PET) of HER2-Positive Breast Cancer Xenografts in Mice with 89Zr-Labeled TrastuzumabDM1 (T-DM1) – Comparison with 89Zr-Labeled Trastuzumab Noor Al-saden, Karen Lam, Conrad Chan, and Raymond M Reilly Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00392 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 4, 2018
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Molecular Pharmaceutics
Positron-Emission Tomography (PET) of HER2-Positive Breast Cancer Xenografts in Mice with
89
Zr-Labeled Trastuzumab-DM1
(T-DM1) – Comparison with 89Zr-Labeled Trastuzumab
Noor Al-saden, † Karen Lam, † Conrad Chan, † and Raymond M. Reilly *,†,§, ¥
†
Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada §
Department of Medical Imaging, University of Toronto, 263 McCaul Street, Toronto, ON M5T 1W7, Canada
¶
Toronto General Research Institute and Joint Department of Medical Imaging, University Health Network, 200 Elizabeth Street, Toronto, ON M5G 2C4, Canada.
Footline: PET Imaging of Breast Cancer with 89Zr-DFO-T-DM1 * Corresponding Author: Raymond M Reilly, Ph.D. Leslie Dan Faculty of Pharmacy, University of Toronto 144 College Street, Toronto, ON M5S 3M2, Canada Tel: (416) 946-5522; Fax: (416) 978-8511; Email:
[email protected] Word Count: 8389
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GRAPHICAL ABSTRACT
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Molecular Pharmaceutics
ABSTRACT: Our aim was to synthesize
89
Zr-labeled trastuzumab-emtansine (89Zr-
DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89Zr-DFO-T-DM1 with 89Zr-DFO-trastuzumab. TDM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after reaction with FeCl3.
SDS-PAGE
and
SE-HPLC
demonstrated
a
pure
and
homogeneous
immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with
89
Zr at a specific activity of 0.55 MBq/µg in 2 M Na2CO3/0.5 M HEPES
buffer, pH 7.0 at RT for 60-90 mins. Labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89Zr-DFO-T-DM1 exhibited high affinity HER2 binding (Kd = 3.7 ± 0.4 nM) that was not significantly different than 89Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P=0.06). The optimal time for tumor imaging with 89Zr-DFO-T-DM1 was 96 h post-injection (p.i.) in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDAMB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+) or MDA-MB-468 (HER2 0) human BC xenografts injected with 89Zr-DFO-T-DM1 (10 µg, 5.2 MBq). All tumors were visualized by microPET/CT but the tumor intensity was greatest for BT-474 and MDAMB-231/H2N xenografts. Tumor uptake of
89
Zr-DFO-T-DM1 was 4.1-fold significantly
higher than 89Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ±
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4.3%ID/g vs. 10.6 ± 5.4 %ID/g, respectively; P
