Abstract 3912: Pre-clinical developments of the G3 Designed ankyrin repeat protein (DARPin) for in vivo assessment of HER2 expression .
Authors: Goldstein Robert , Tolner Berend , Leyton Julius , Livanos Maria , Bhavsar Gaurav , Vigor Kim , Nagy Gabriela , Mather Steve , Plückthun Andreas , Sosabowski Jane , Meyer Tim , Chester Kerry 
Published: August 2014

Abstract
Background:
Breast cancer HER2 molecular imaging can potentially identify disease relapse, inform treatment decisions and assess treatment responses. Molecular imaging relies upon achieving high tumour:blood and tumour:normal tissue ratios. The G3 DARPin is a small protein with picomolar affinity for HER2, based on the ankyrin repeat scaffold that is expressed in humans. The hexahistidine (His6) tagged G3 DARPin labelled with 99mTc(CO)3 can image HER2+ SK-OV-3 tumours [Zahnd et al. Cancer Res 2010;70:1595-605].
Alteration of the His6 tag to a negatively charged and hydrophilic histidine-glutamate (HE)3 tag can reduce background liver uptake, while enabling tag mediated purification by immobilised metal affinity chromatography [Hofstrom et al. J Med Chem 2011;54;3817-26]. We hypothesized that the biodistribution of 111In and 125I G3 DARPin could be optimised by altering the N-terminal domain.
Methods:
His6, HE3 and untagged G3 were produced in E. coli and or P. pastoris and labelled directly with 125I or with DOTA via a C-terminal cysteine for 111In. BALB/c mice were injected with 0.3 MBq of 111In or 125I G3. The optimal G3 construct was assessed with 111In and 125I in HER2+ human breast tumour (BT474)-bearing mice.
Results:
Biodistribution of the DARPins was evaluated in BALB/c mice at 4 and 24 h. Results showed that 111In-HE3-G3 had lower or similar uptake to 111In-His6-G3 and 111In-untagged-G3 in 11 different normal tissues tested. Superiority of HE3-G3 for normal tissue uptake was also observed when the DARPins were labelled with 125I.
HE3-G3 was assessed in HER2+ tumour-bearing mice. The tumour uptake for 125I-HE3-G3 was approximately 2 fold higher than 111In-HE3-G3 at 4 h. However, 111In-HE3-G3 tumour uptake was better maintained, so that by 24 h 111In-HE3-G3 tumour uptake was approximately 1.5 fold higher than 125I-HE3-G3. Normal tissue uptake was generally lower for 111In-HE3-G3 than 125I-HE3-G3 at 4 h, except in the kidneys which were higher for 111In-HE3-G3 throughout. At 24 h, the differences in normal tissue uptake between 111In-HE3-G3 and125I-HE3-G3 were smaller. 111In-HE3-G3 had faster serum clearance than 125I-HE3-G3, resulting in higher normal tissue:blood ratios for all assessed tissues except stomach. As a consequence, the tumour:blood ratios for 111In-HE3-G3 were the most impressive, &gt; 150:1 at 4 h and &gt; 300:1 at 24 h . 111In-HE3-G3 microSPECT/CT imaging demonstrated tumour uptake at 2 and 4 h.
Conclusions:
N-terminal tags effect tissue biodistribution of G3. HE3-G3 radiolabelled with 111In and 125I had lower uptake in normal tissues compared to untagged or His6 tagged G3. 111In-HE3-G3 achieved and maintained the highest tumour:blood ratios over 24 h. Based on its superiority, development will focus on the radiolabelled C-terminal cysteine DOTA conjugated HE3-G3 for SPECT and PET HER2 imaging.
Citation Format: Robert Goldstein, Berend Tolner, Julius Leyton, Maria Livanos, Gaurav Bhavsar, Kim Vigor, Gabriela Nagy, Steve Mather, Andreas Plückthun, Jane Sosabowski, Tim Meyer, Kerry Chester. Pre-clinical developments of the G3 Designed ankyrin repeat protein (DARPin) for in vivo assessment of HER2 expression . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3912. doi:10.1158/1538-7445.AM2013-3912