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64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR –Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents Luke (Yongkyu) Kwon, Deborah A Scollard, and Raymond M Reilly Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00963 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017
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Molecular Pharmaceutics
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Cu-Labeled Trastuzumab Fab-PEG24-EGF
Radioimmunoconjugates Bispecific for HER2 and EGFR – Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents Luke Yongkyu Kwon1, Deborah A Scollard2 and Raymond M Reilly1,3,4,5 1
Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada; 2
STTARR Innovation Centre, University Health Network, Toronto, Ontario, Canada; 3
Department of Medical Imaging, University of Toronto, Toronto, ON, Canada;
Toronto General Research Institute; and
5
4
Joint Department of Medical Imaging,
University Health Network, Toronto, ON, Canada.
AUTHOR INFORMATION * Corresponding author: Raymond M. Reilly, Ph.D., Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON, Canada, M5S 3M2. Tel: 1(416)-946-5522. Fax: (416)-978-8511. E-mail:
[email protected] Revision: December 23, 2016
Conflict of interest statement: The authors declare no competing financial interest
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ABSTRACT Heterodimerization of EGFR with HER2 co-expressed in breast cancer (BC) promotes tumor growth and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct
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Cu-labeled bispecific radioimmunoconjugates (bsRIC)
composed of trastuzumab Fab which binds HER2 linked through a polyethylene glycol (PEG24) spacer to EGF, and compare their pharmacokinetic, biodistribution and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking malemide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFRmod/HER2low),
MDA-MB-468
EGFRhigh/HER2neg),
MDA-MB-231-H2N
(EGFRmod/HER2mod), and SKOV3 (EGFRlow/HER2high) cells by competition and saturation cell binding assays to estimate the dissociation constant (Kd). The elimination of the 64Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs from the blood of Balb/c mice was compared to monospecific
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Cu-NOTA-trastuzumab Fab and
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Cu-NOTA-EGF.
MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDAMB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2.
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Cu-labeled bsRICs
incorporating the PEG24 spacer were eliminated more slowly from the blood than 64CubsRICs without the PEG spacer, and were cleared much more slowly than 64Cu-NOTA-
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Molecular Pharmaceutics
Fab or 64Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of NOTA-Fab-PEG24-EGF (4.9 ± 0.4 %ID/g) than P
