Human epidermal growth factor receptor 3 (HER3) is involved in the progression of various cancers and in resistance to therapies targeting the HER family. In vivo imaging of HER3 expression would enable patient stratification for anti-HER3 immunotherapy. Key challenges with HER3-targeting are the relatively low expression in HER3-positive tumours and HER3 expression in normal tissues. The use of positron-emission tomography (PET) provides advantages of high resolution, sensitivity and quantification accuracy compared to SPECT. Affibody molecules, imaging probes based on a non-immunoglobulin scaffold, provide high imaging contrast shortly after injection. The aim of this study was to evaluate feasibility of PET imaging of HER3 expression using 68Ga-labeled affibody molecules. The anti-HER3 affibody molecule HEHEHE-Z08698-NOTA was successfully labelled with 68Ga with high yield, purity and stability. The agent bound specifically to HER3-expressing cancer cells in vitro and in vivo. At 3 h pi, uptake of 68Ga-HEHEHE-Z08698-NOTA was significantly higher in xenografts with high HER3 expression (BT474, BxPC-3) than in xenografts with low HER3 expression (A431). In xenografts with high expression, tumour-to-blood ratios were >20, tumour-to-muscle >15, and tumour-to-bone >7. HER3-positive xenografts were visualised using microPET 3 h pi. In conclusion, PET imaging of HER3 expression is feasible using 68Ga-HEHEHE-Z08698-NOTA shortly after administration.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a positron-emitting radionuclide 89Zr. The 89Zr-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160±60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, 89Zr-DFO-ZEGFR:2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6±0.5% ID/g and tumour-to-blood ratio of 3.7±0.6 at 24 h after injection. 89Zr-DFO-ZEGFR:2377 provides higher tumour-to-organ ratios than anti-EGFR antibody 89Zr-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using 89Zr-DFO-ZEGFR:2377 at both 3 and 24 h after injection. In conclusion, 89Zr-DFO-ZEGFR:2377 is a potential probe for PET imaging of EGFR-expression in vivo.

Imaging of HER3-expressing xenografts in mice using a (99m)Tc(CO) 3-HEHEHE-Z HER3 08699 affibody molecule. Tc(CO) 3 -HEHEHE-Z 08699 was studied over time in mice bearing HER3-expressing xenografts. European Journal of Nuclear Medicine and MolecularResults. HEHEHE-Z 08699 was labeled with 99m Tc(CO) 3 with an isolated yield of >80% and a purity of >99%. Binding of 99m Tc(CO) 3 -HEHEHE-Z 08699 was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of receptor/affibody molecule complex (70% cell associated radioactivity was internalized after 24 h). Tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor mediated uptake was also found in liver, lung, stomach, intestine, and salivary glands.At 4 h pi, tumor-to-blood ratios were 7±3 for BT474, and 6±2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. Conclusions. The results of this study suggest feasibility of HER3-imaging in malignant tumors using affibody molecules.

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with 57Co (T1/2 = 271.8 d), 55Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57Co was used in animal studies. Both 57Co-DOTA-ZEGFR:2377 and 68Ga-DOTA-ZEGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-ZEGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57Co-DOTA-ZEGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68Ga-DOTA-ZEGFR:2377. The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-ZEGFR:2377 and further development should concentrate on this radionuclide as a label.

Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

X-ray fluorescence computed tomography (XFCT) with nanoparticles (NPs) as contrast agents shows potential for molecular biomedical imaging with higher spatial resolution than present methods. To date the technique has been demonstrated on phantoms and mice, however, parameters such as radiation dose, exposure times and sensitivity have not yet allowed for high-spatial-resolutionin vivo longitudinal imaging, i.e., imaging of the same animal at different time points. Here we show in vivo XFCT with spatial resolution in the 200-400 µm range in a proof-of-principle longitudinal study where mice are imaged five times each during an eight-week period following tail-vein injection of NPs. We rely on a 24 keV x-ray pencil-beam-based excitation of in-house-synthesized molybdenum oxide NPs (MoO 2) to provide the high signal-to-background x-ray fluorescence detection necessary for XFCT imaging with low radiation dose and short exposure times. We quantify the uptake and clearance of NPs in vivo through imaging, and monitor animal well-being over the course of the study with support from histology and DNA stability analysis to assess the impact of x-ray exposure and NPs on animal welfare. We conclude that the presented imaging arrangement has potential for in vivo longitudinal studies, putting emphasis on designing biocompatible NPs as the future focus for active-targeting preclinical XFCT.

Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7 kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium-111 (111In) and assessed in vitro and in vivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. In vitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT-474 breast carcinoma cells. In mice bearing BT-474 xenografts, the tumor uptake of the two conjugates was receptor-specific. Direct in vivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12 ± 3 vs. 8 ± 1, 4 h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N′,N′′-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)−4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z08698 and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged 111In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.