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

Robert Goldstein,Stephen Mather,Berend Tolner,Jane Sosabowski,Mohammed Rashid,Jenny Yeung,Tim Meyer,Gabriela Nagy-Davidescu,Enrique Miranda,Kerry Chester,Gaurav Bhavsar,Julius Leyton,Andreas Plückthun,Maria Livanos,Kim Vigor

doi:10.1007/s00259-014-2940-2

Abstract

PurposeHuman 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.MethodsG3 DARPins tagged with hexahistidine (His6) 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.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-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.ConclusionRadiolabelled 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 materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.

Highlights

Overexpression of human epidermal growth factor receptor-2 (HER2) enhances signal transduction through the PI3K/Akt and the Ras/Raf/MEK/MAPK pathways, enabling cancer cell proliferation and survival [1]
A competition assay showed that the His6-G3 labelled with cold indium and cold iodine, (HE)3-G3 and untagged G3 all competed with [99mTc(CO)3]+-G3 designed ankyrin repeat protein (DARPin)-His6 for binding to HER2-positive human breast cancer cells (BT474)
Flow cytometry demonstrated that G3 DARPins labelled with cold indium and cold iodine bound to HER2-positive human breast cancer cells (BT474) and HER2-positive human gastrooesophageal junction (OE-19) adenocarcinoma cells, but did not bind to HER2-negative human breast adenocarcinoma cells (MDA-MB-468)

Summary

Introduction

Overexpression of human epidermal growth factor receptor-2 (HER2) enhances signal transduction through the PI3K/Akt and the Ras/Raf/MEK/MAPK pathways, enabling cancer cell proliferation and survival [1]. Patients are selected for anti-HER2 therapy based on histological analysis, using immunohistochemistry or fluorescence in situ hybridization (FISH) of biopsied or surgically resected tissues [5]. These analyses are limited by the use of single-site and single time-point sampling, and they fail to provide information about heterogeneity of expression or changes in expression that occur over time [6]. The impact of HER2 misclassification on clinical outcomes is unclear, 20 – 30 % of patients with HER2-positive advanced breast cancer do not respond to first-line treatment combinations, despite advances in anti-HER2 therapies [10]. Some patients with breast cancer histologically classified as HER2-negative benefit from adjuvant anti-HER2 therapy, casting doubt on their classification [11]

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