2013 NCRI Cancer Conference

3 - 6 November 2013
The BT Convention Centre Liverpool UK


Developing Designed Ankyrin Repeat Proteins (DARPins) for HER2 imaging

Robert Goldstein1, Tim Meyer1, Berend Tolner1, Kerry Chester1, Maria Livanos1, Kim Vigor1, Gaurav Bhavsar1, Jane Sosabowski2, Steve Mather2, Julius Leyton2, Andreas Plückthun3, Gabriela Nagy3
1UCL Cancer Institute, London, UK, 2Barts Cancer Institute, London, UK, 3University of Zurich, Zurich, UK


HER2 imaging can potentially assist in clinical management. The G3 DARPin, a small protein based on human ankyrin repeats1, was developed as a novel imaging agent. The DARPin has picomolar affinity for HER2 and is generated as a recombinant protein with N-terminal tags for purification. The bio-distribution of G3 was tested to evaluate the influence of tags and choice of radiolabel.


The DARPin was generated in P. pastoris, and purified with either a hexahistidine (His6 tag) or a more negatively charged hydrophilic histidine-glutamate (HE3 tag). Untagged G3 was used a control. The purified proteins were labelled with 125I (using iodogen) or 111In (via site-specific attachment of DOTA). Bio-distribution was assessed in BALB/c mice or nude mice bearing HER2+ human (BT474) breast tumours.


111In-His6-G3 and untagged 111In -G3 had significantly higher liver uptake than 111In-HE3-G3 at 4 h (p=0.001) and 24 h (p=0.001 and p=0.002 respectively) post administration. Superiority of the HE3 tag was also observed with 125I-radiolabelled-G3. HE3-G3 was therefore taken forward for testing in HER2+ tumour-bearing mice.

Tumour uptake of 125I-HE3-G3 and 111In-HE3-G3 were similar at 4 h butthe 111In-HE3-G3 tumours retained more radioactivity over time, resulting in ~3-fold higher than value for 111In-HE3-G3 (residualizing radionuclide) than 125I-HE3-G3 (non-residualizing) at 24 h. 111In-HE3-G3 also had faster serum clearance than 125I-HE3-G3, resulting in higher normal tissue:blood ratios for all assessed tissues (except stomach). Tumour:blood ratios of >300:1 were achieved at 24 h whilst maintaining ~ 8% of the injected radioactivity/g of tumour. Pre-administration of trastuzumab did not alter HER2 tumour 111In-HE3-G3 uptake. HER2+ tumour imaging was demonstrated by micro SPECT/CT.


The HE3-G3 was superior to His-G3 or untagged G3 and 111In was superior to 125I. Clinical development will focus on chelate radiolabelled HE3-G3 for SPECT and PET HER2 imaging.


This study was supported by: European Framework FP7 IMAGINT Project (EC GRANT Agreement No. 259881), The Breast Cancer Campaign, Cancer Research UK (CRUK); The Experimental Cancer Medicine Centre (ECMC) and The UCLH/UCL Department of Health’s NIHR Biomedical Research Centre (BRC) funding scheme.


1. Zahnd et al. Cancer Res 2010;70:1595-605.