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.

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.

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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, > 150:1 at 4 h and > 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