Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [125I]I-para-iodobenzoate (PIB) label and a residualizing [99mTc]Tc(CO)3 label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [125I]I-PIB-Ec1 and [99mTc]Tc(CO)3-Ec1 in TNBC tumors. [125I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [99mTc]Tc(CO)3-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.

The use of radiolabelled antibodies was proposed in 1970s for staging of malignant tumours. Intensive research established chemistry for radiolabelling of proteins and understanding of factors determining biodistribution and targeting properties. The use of radioimmunodetection for staging of cancer was not established as common practice due to approval and widespread use of [18F]-FDG, which provided a more general diagnostic use than antibodies or their fragments. Expanded application of antibody-based therapeutics renewed the interest in radiolabelled antibodies. RadioimmunoPET emerged as a powerful tool for evaluation of pharmacokinetics of and target engagement by biotherapeutics. In addition to monoclonal antibodies, new radiolabelled engineered proteins have recently appeared, offering high-contrast imaging of expression of therapeutic molecular targets in tumours shortly after injection. This creates preconditions for noninvasive determination of a target expression level and stratification of patients for targeted therapies. Radiolabelled proteins hold great promise to play an important role in development and implementation of personalised targeted treatment of malignant tumours. This article provides an overview of biodistribution and tumour-seeking features of major classes of targeting proteins currently utilized for molecular imaging. Such information might be useful for researchers entering the field of the protein-based radionuclide molecular imaging.

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.

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 (89)Zr. The (89)Zr-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, (89)Zr-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. (89)Zr-DFO-ZEGFR:2377 provides higher tumour-to-organ ratios than anti-EGFR antibody (89)Zr-DFO-cetuximab at 48 h after injection. EGFR‑expressing tumours were clearly visualized by microPET using (89)Zr-DFO-ZEGFR:2377 at both 3 and 24 h after injection. In conclusion, 8(9)Zr-DFO-ZEGFR:2377 is a potential probe for PET imaging of EGFR-expression in vivo.

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with 111In and characterized in vitro. Tumor-targeting properties of [111In]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [99mTc]Tc(CO)3-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [111In]In-DTPA-G250(Fab')2, in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the 99mTc-labeled parental variant, [111In]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [111In]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [111In]In-G250(Fab')2. In conclusion, [111In]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

Evaluation of human epidermal growth factor receptor 2 (HER2) expression levels in breast and gastroesophageal cancer is used for the stratification of patients for HER2‑targeting therapies. The use of radionuclide molecular imaging may facilitate such evaluation in a non‑invasive way. Designed ankyrin repeat proteins (DARPins) are engineered scaffold proteins with high potential as probes for radionuclide molecular imaging. DARPin G3 binds with high affinity to HER2 and may be used to visualize this important therapeutic target. Studies on other engineered scaffold proteins have demonstrated that selection of the optimal labeling approach improves the sensitivity and specificity of radionuclide imaging. The present study compared two methods of labeling G3, direct and indirect radioiodination, to select an approach providing the best imaging contrast. G3‑H6 was labeled with iodine‑124, iodine‑125 and iodine‑131 using a direct method. A novel construct bearing a C‑terminal cysteine, G3‑GGGC, was site‑specifically labeled using [125I]I‑iodo‑[(4‑hydroxyphenyl)ethyl]maleimide (HPEM). The two radiolabeled G3 variants preserved binding specificity and high affinity to HER2‑expressing cells. The specificity of tumor targeting in vivo was demonstrated. Biodistribution comparison of [131I]I‑G3‑H6 and [125I]I‑HPEM‑G3‑GGGC in mice, bearing HER2‑expressing SKOV3 xenografts, demonstrated an appreciable contribution of hepatobiliary excretion to the clearance of [125I]I‑HPEM‑G3‑GGGC and a decreased tumor uptake compared to [131I]I‑G3‑H6. The direct label provided higher tumor‑to‑blood and tumor‑to‑organ ratios compared with the indirect label at 4 h post‑injection. The feasibility of high contrast PET/CT imaging of HER2 expression in SKOV3 xenografts in mice using [124I]I‑G3‑H6 was demonstrated. In conclusion, direct radioiodination is the preferable approach for labeling DARPin G3 with iodine‑123 and iodine‑124 for clinical single photon emission computed tomography and positron emission tomography imaging.

Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide 68Ga. Stability, specificity of binding to EGFR-expressing cells, and processing of [68Ga]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [68Ga]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [89Zr]Zr-DFO-ZEGFR:2377.

The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide 99mTc should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with 99mTc using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, 99mTc-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that 99mTc-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6±1 and 2.5±0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8±0.4 and 8±3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of 99mTc-labeled ZEGFR:2377 for imaging of EGFR in vivo.

Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient. A previous study demonstrated that affibody molecule [68Ga]Ga-DFO-ZEGFR:2377 permits specific positron-emission tomography (PET) imaging of EGFR expression in xenografts at 3 h after injection. We anticipated that imaging at 24 h after injection would provide higher contrast, but this is prevented by the short half-life of 68Ga (67.6 min). Here, we therefore tested the hypothesis that the use of the non-conventional long-lived positron emitter 66Ga (T1/2 = 9.49 h, β+ = 56.5%) would permit imaging with higher contrast. 66Ga was produced by the 66Zn(p,n)66Ga nuclear reaction and DFO-ZEGFR:2377 was efficiently labelled with 66Ga with preserved binding specificity in vitro and in vivo. At 24 h after injection, [66Ga]Ga-DFO-ZEGFR:2377 provided 3.9-fold higher tumor-to-blood ratio and 2.3-fold higher tumor-to-liver ratio than [68Ga]Ga-DFO-ZEGFR:2377 at 3 h after injection. At the same time point, [66Ga]Ga-DFO-ZEGFR:2377 provided 1.8-fold higher tumor-to-blood ratio, 3-fold higher tumor-to-liver ratio, 1.9-fold higher tumor-to-muscle ratio and 2.3-fold higher tumor-to-bone ratio than [89Zr]Zr-DFO-ZEGFR:2377. Biodistribution data were confirmed by whole body PET combined with magnetic resonance imaging (PET/MRI). The use of the positron emitter 66Ga for labelling of DFO-ZEGFR:2377 permits PET imaging of EGFR expression at 24 h after injection and improves imaging contrast.

Zirconium-89 is an emerging radionuclide for positron emission tomography (PET) especially for biomolecules with slow pharmacokinetics as due to its longer half-life, in comparison to fluorine-18 and gallium-68, imaging at late time points is feasible. Desferrioxamine B (DFO), a linear bifunctional chelator (BFC) is mostly used for this radionuclide so far but shows limitations regarding stability. Our group recently reported on fusarinine C (FSC) with similar zirconium-89 complexing properties but potentially higher stability related to its cyclic structure. This study was designed to compare FSC and DFO head-to-head as bifunctional chelators for 89Zr-radiolabeled EGFR-targeting ZEGFR:2377 affibody bioconjugates.

Albumin binding domain-Derived Affinity ProTeins (ADAPTs) are small (5 kDa) engineered scaffold proteins that are promising targeting agents for radionuclide-based imaging. A recent clinical study has demonstrated that radiolabeled ADAPTs can efficiently visualize human epidermal growth factor receptor 2 (HER2) expression in breast cancer using SPECT imaging. However, the use of ADAPTs directly labeled with radiometals for targeted radionuclide therapy is limited by their high reabsorption and prolonged retention of activity in kidneys. In this study, we investigated whether a co-injection of lysine or gelofusin, commonly used for reduction of renal uptake of radiolabeled peptides in clinics, would reduce the renal uptake of [99mTc]Tc(CO)3-ADAPT6 in NMRI mice. In order to better understand the mechanism behind the reabsorption of [99mTc]Tc(CO)3-ADAPT6, we included several compounds that act on various parts of the reabsorption system in kidneys. Administration of gelofusine, lysine, probenecid, furosemide, mannitol, or colchicine did not change the uptake of [99mTc]Tc(CO)3-ADAPT6 in kidneys. Sodium maleate reduced the uptake of [99mTc]Tc(CO)3-ADAPT6 to ca. 25% of the uptake in the control, a high dose of fructose (50 mmol/kg) reduced the uptake by ca. two-fold. However, a lower dose (20 mmol/kg) had no effect. These results indicate that common clinical strategies are not effective for reduction of kidney uptake of [99mTc]Tc(CO)3-ADAPT6 and that other strategies for reduction of activity uptake or retention in kidneys should be investigated for ADAPT6.

ABD-Derived Affinity Proteins (ADAPTs) is a novel class of engineered scaffold proteins derived from an albumin-binding domain of protein G. The use of ADAPT6 derivatives as targeting moiety have provided excellent preclinical radionuclide imaging of human epidermal growth factor 2 (HER2) tumor xenografts. Previous studies have demonstrated that selection of nuclide and chelator for its conjugation has an appreciable effect on imaging properties of scaffold proteins. In this study we performed a comparative evaluation of the anti-HER2 ADAPT having an aspartate-glutamate-alanine-valine-aspartate-alanine-asparagine-serine (DEAVDANS) N-terminal sequence and labeled at C-terminus with 99mTc using a cysteine-containing peptide based chelator, glycine-serine-serine-cysteine (GSSC), and a similar variant labeled with 111In using a maleimido derivative of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. Both 99mTc-DEAVDANS-ADAPT6-GSSC and 111In-DEAVDANS-ADAPT6-GSSC-DOTA accumulated specifically in HER2-expressing SKOV3 xenografts. The tumor uptake of both variants did not differ significantly and average values were in the range of 19-21%ID/g. However, there was an appreciable variation in uptake of conjugates in normal tissues that resulted in a notable difference in the tumor-to-organ ratios. The 111In-DOTA label provided 2-6 fold higher tumor-to-organ ratios than 99mTc-GSSC and is therefore the preferable label for ADAPTs.

Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of 99mTc, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The 99mTc-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules.

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARPin G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)3) of histidine-containing tags would influence the biodistribution of [99mTc]Tc(CO)3-labeled DARPin G3. To test the hypothesis, G3 variants containing tags at N-terminus (H6-G3 and (HE)3-G3) or at C-terminus (G3-H6 and G3-(HE)3) were labeled with [99mTc]Tc(CO)3. Labeling yield, label stability, specificity and affinity of the binding to HER2, biodistribution and tumor targeting properties of these variants were compared side-by-side. There was no substantial influence of position and composition of the tags on binding of [99mTc]Tc(CO)3-labeled variants to HER2. The specificity of HER2 targeting in vivo was confirmed. The tumor uptake in BALB/c nu/nu mice bearing SKOV3 xenografts was similar for all variants. On the opposite, there was a strong influence of the tags on uptake in normal tissues. The tumor-to-liver ratio for [99mTc]Tc(CO)3-(HE)3-G3 was three-fold higher compared to the hexahistidine-tag containing variants. Overall, [99mTc]Tc(CO)3-(HE)3-G3 variant provided the highest tumor-to-lung, tumor-to-liver, tumor-to-bone and tumor-to-muscle ratios, which should improve sensitivity of HER2 imaging in these common metastatic sites.