Abstract 5442: Biodistribution and antitumor efficacy study of novel Her2 targeting DARPins
Authors: Raguin Olivier , Leblanc Marie , Collin Bertrand , Oudot Alexandra , Mirjolet Jean-François , Fiedler Ulrike , Dolado Ignacio 
Published: May 2015

Abstract
Introduction:
DARPins are genetically engineered small-sized proteins exhibiting high affinity for target proteins. The epithelial growth factor receptor 2 (Her2/neu) is a target of interest for targeted therapies as it is overexpressed in epithelial tumors such as breast and gastric cancers. Antitumor activity and biodistribution of novel Her2-targeting DARPins were evaluated in mice bearing Her2-positive human breast tumors.
Materials and methods:
Antitumor activity and biodistribution studies were performed in female Balb/c Nude mice bearing subcutaneous BT-474 human breast tumors. Mice were treated intravenously (IV) every 3 days with five different DARPins at 20 or 35 mg/kg and antitumor activity was evaluated by measuring tumor growth twice weekly. For the biodistribution study, DARPins were radiolabelled with technetium-99m (99mTc) using the IsoLink procedure. Quality control of the radiolabelled DARPins was performed by size exclusion chromatography, ELISA assay, and binding to tumor cells. 99mTc-radiolabelled DARPins were injected IV to mice at 23-25 MBq/mouse. 4 and 24 hours post-injection, animals were euthanized for blood, tumor and organ collection. Radioactivity in each sample was measured and normalized to sample weight and injected dose (% ID/g). The depth of tumor penetration of 99mTc-radiolabelled DARPins was also assessed by planar autoradiography imaging.
Results:
All five DARPins displayed a significant antitumor activity. Four DARPins induced BT-474 tumor regression while the fifth led to BT-474 tumor stasis. All DARPins were efficiently radiolabelled with 99mTc to radiochemical purities within 50% to 90%, and they all retained the ability to fully bind Her2. The five DARPins displayed a comparable distribution pattern. Radioactivity uptake in blood ranged from 25-30% and 8-10 % ID/g at 4 and 24 hours post-injection, respectively. Accumulation in tumors increased from 6-10% ID/g at 4 hours to 12-20% ID/g at 24 hours. All 99mTc-radiolabelled DARPins were excreted through the liver and kidneys and radioactivity uptake in both organs, respectively, ranged from 7-14% ID/g and 4-8% ID/g at 24 hours post-injection. Planar autoradiography imaging revealed that the depth of penetration of DARPinsinto the tumor was &gt; 2 mm as soon as 4 hours post-injection.
Conclusions:The five anti-Her2 DARPins investigated efficiently targeted BT-474 tumors and significantly inhibited their growth, making them attractive as new therapeutic agents for the treatment of Her2-positive cancers. Their easy radiolabelling with 99mTc also makes them attractive tools for imaging with diagnostic and predictive purposes, for instance to follow the modulation of Her2 expression during therapy.
Citation Format: Olivier Raguin, Marie Leblanc, Bertrand Collin, Alexandra Oudot, Jean-François Mirjolet, Ulrike Fiedler, Ignacio Dolado. Biodistribution and antitumor efficacy study of novel Her2 targeting DARPins. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5442. doi:10.1158/1538-7445.AM2014-5442