Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

Abstract

Purpose

Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.

Methods

G3 DARPins tagged with hexahistidine (His ₆) or with histidine glutamate (HE) ₃ and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with ¹²⁵I, or with ¹¹¹In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE) ₃-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.

Results

For both isotopes, (HE) ₃-G3 had significantly lower liver uptake than His ₆-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His ₆-G3 and untagged G3. (HE) ₃-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from ¹¹¹In-(HE) ₃-G3 was better maintained in tumours and cleared faster from serum than radioactivity from ¹²⁵I-(HE) ₃-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, ¹¹¹In-labelled and ¹²⁵I-labelled (HE) ₃-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with ¹¹¹In-(HE) ₃-G3. Preadministration of trastuzumab did not affect the uptake of (HE) ₃-G3 by HER2-positive tumours.

Conclusion

Radiolabelled DARPin (HE) ₃-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.

Electronic supplementary material

The online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.