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Increasing the net negative charge by replacement of DOTA chelator with DOTAGA improves the biodistribution of radiolabeled second-generation synthetic Affibody molecules Kristina Westerlund, Hadis Honarvar, Emily Norrström, Joanna Strand, Mitran Bogdan, Anna Orlova, Amelie Eriksson Karlström, and Vladimir Tolmachev Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00089 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016
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Molecular Pharmaceutics
Increasing the net negative charge by replacement of DOTA chelator with DOTAGA improves the biodistribution of radiolabeled second-generation synthetic Affibody molecules
Kristina Westerlund§a, Hadis Honarvar§b, Emily Norrströma, Joanna Strandb, Bogdan Mitranc, Anna Orlovac, Amelie Eriksson Karlström*a, Vladimir Tolmachevb.
§
These authors contributed equally
a
Division of Protein Technology, School of Biotechnology, KTH Royal Institute of
Technology, Stockholm, Sweden b
Institute for Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden
c
Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala University, Uppsala,
Sweden
* Corresponding author E-mail:
[email protected] Phone: +46-8-5537 8333 Fax: +46-8-5537 8481
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Molecular Pharmaceutics
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ABSTRACT A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are non-immunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the Nterminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 ZHER2:2891 Affibody molecule was synthesized and labeled with 111In and 68Ga using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of ZHER2:2891 forming an amide bond. Labeling DOTAGA-ZHER2:2891 and DOTA-ZHER2:2891 with 68Ga and 111In resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the 111In- and 68Galabeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h post injection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The 111In-labeled conjugates showed more rapid blood clearance than the 68Galabeled conjugates. The most pronounced influence of the chelators was found when they were labeled with 68Ga. The DOTAGA chelator gave significantly higher tumor-to-blood (61±6 vs 23±5, p
