Radionuclide molecular imaging of human epidermal growth factor type 2 (HER2) expression may enable a non-invasive discrimination between HER2-positive and HER2-negative breast cancers for stratification of patients for HER2-targeted treatments. DARPin G3 is a small (molecular weigh 14 kDa) scaffold protein with picomolar affinity to HER2. The aim of this first-in-human study was to evaluate the safety, biodistribution and dosimetry of ^99mTc-(HE)₃-G3. Methods: Three cohorts of patients with primary breast cancer (each including at least 4 patients with HER2-negative and 5 patients with HER2-positive tumors) were injected with either 1000, 2000 or 3000 µg of ^99mTc-(HE)₃-G3 (287±170 MBq). Whole-body planar imaging followed by SPECT was performed at 2, 4, 6 and 24 h after injection. Vital signs and possible side effects were monitored during imaging and up to 7 days after injection. Results: All injections were well tolerated. No side effects were observed. The results of blood and urine analyses did not differ before and after studies. ^99mTc-(HE)₃-G3 cleared rapidly from the blood. The highest uptake was detected in the kidneys and liver followed by the lungs, breasts and small intestinal content. The hepatic uptake after injecting with 2000 or 3000 µg was significantly (p<0.05) lower than the uptake after injecting with 1000 µg. Effective doses did not differ significantly between cohorts (average 0.011± 0.004 mSv/MBq). Tumor-to-contralateral site ratios for HER-positive tumors were significantly (p< 0.05) higher than for HER2-negative at 2 and 4 h after injection. Conclusion: Imaging of HER2 expression using ^99mTc-(HE)₃-G3 is safe, well-tolerated and provides a low absorbed dose burden on patients. This imaging enables discerning HER2-positive and HER2-negative breast cancer. Phase I study data justifies further clinical development of ^99mTc-(HE)₃-G3.

Gastrin‐releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin‐derived GRPR‐antagonist PEG₂‐RM26 for labeling with ¹⁷⁷Lu and further determined the effect of treatment with ¹⁷⁷Lu‐labeled peptide alone or in combination with the anti‐HER2 antibody trastuzumab in a murine model. The PEG₂‐RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide‐chelator conjugates were labeled with ¹⁷⁷Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC‐3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA‐PEG₂‐RM26, (C) ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26, (D) trastuzumab or (E) ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 in combination with trastuzumab. ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/μmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (K_D = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment‐related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four ¹⁷⁷Lu‐labeled PEG₂‐RM26 analogs, we concluded that ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 was the most promising analog for TRT. Radiotherapy using ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti‐HER2 therapy additionally improved survival.

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.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.

High expression of human epidermal growth factor receptor 2 (HER2) in breast and gastroesophageal carcinomas is a predictive biomarker for treatment using HER2-targeted therapeutics (antibodies trastuzumab and pertuzumab, antibody-drug conjugate trastuzumab DM1, and tyrosine kinase inhibitor lapatinib). Radionuclide molecular imaging of HER2 expression might permit stratification of patients for HER2-targeting therapies. In this study, we evaluated a new HER2-imaging probe based on the designed ankyrin repeat protein (DARPin) 9_29. DARPin 9_29 was labeled with iodine-125 by direct radioiodination and with [^99mTc]Tc(CO)₃ using the C-terminal hexahistidine tag. DARPin 9_29 preserved high specificity and affinity of binding to HER2-expressing cells after labeling. Uptake of [¹²⁵I]I-DARPin 9_29 and [^99mTc]Tc(CO)₃-DARPin 9_29 in HER2-positive SKOV-3 xenografts in mice at 6 h after injection was 3.4 ± 0.7 %ID/g and 2.9 ± 0.7 %ID/g, respectively. This was significantly (p<0.00005) higher than the uptake of the same probes in HER2-negative Ramos lymphoma xenografts, 0.22 ± 0.09 %ID/g and 0.30 ± 0.05 %ID/g, respectively. Retention of [¹²⁵I]I-DARPin 9_29 in the lung, liver, spleen, and kidneys was appreciably lower compared with [^99mTc]Tc(CO)₃-DARPin 9_29, which resulted in significantly (p<0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by SPECT/CT imaging. In conclusion, radioiodine is a preferable label for DARPin 9_29.