Developing Designed Ankyrin Repeat Proteins (DARPins) for HER2 imaging
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.