Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. Methods: In total, 20 women with primary or metastatic breast carcinoma (score of 21 or 31 on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with 68 Ga via a NOTA derivative. Administered activities were 53-174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. Results: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. Conclusion: 68 Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial. One in 8 women develops breast cancer, and it remains the second leading cause of cancer death in women. Identification of cancer subtypes based on biologic markers has led to the introduction of targeted therapies, with improved survival and morbidity. Besides hormone receptor expression, human epidermal growth factor receptor 2 (HER2) is used for breast cancer classification. Breast cancers with HER2 overexpression in primary or metastatic sites will benefit from HER2-targeted therapie...

Nanobodies are the smallest fully functional antigen-binding antibody fragments possessing ideal properties as probes for molecular imaging. In this study we labeled the anti-human epidermal growth factor receptor type 2 (HER2) Nanobody with 68 Ga via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and assessed its use for HER2 iPET imaging. Methods: The 2Rs15dHis 6 Nanobody and the lead optimized current-good-manufacturing-practice grade analog 2Rs15d were conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) to enable fast and efficient 68 Ga labeling. Biodistribution and PET/CT studies were performed on HER2-positive and -negative tumor xenografts. The effect of injected mass on biodistribution was evaluated. The biodistribution data were extrapolated to calculate radiation dose estimates for the adult female using OLINDA software. A single-dose extended-toxicity study for NOTA2Rs15d was performed on healthy mice up to a dose of 10 mg/kg. Results: Radiolabeling was quantitative (.97%) after 5 min of incubation at room temperature; specific activity was 55-200 MBq/nmol. Biodistribution studies showed fast and specific uptake (percentage injected activity [%IA]) in HER2-positive tumors (3.13 6 0.06 and 4.34 6 0.90 %IA/g for 68 Ga-NOTA-2Rs15dHis 6 and 68 Ga-NOTA-2Rs15d, respectively, at 1 h after injection) and high tumor-to-blood and tumor-to-muscle ratios at 1 h after injection, resulting in high-contrast PET/CT images with high specific tumor uptake. A remarkable finding of the biodistribution studies was that kidney uptake was reduced by 60% for the Nanobody lacking the C-terminal His 6 tag. The injected mass showed an effect on the general biodistribution: a 100-fold increase in NOTA-2Rs15d mass decreased liver uptake from 7.43 6 1.89 to 2.90 6 0.26 %IA/g whereas tumor uptake increased from 2.49 6 0.68 to 4.23 6 0.99 %IA/g. The calculated effective dose, based on extrapolation of mouse data, was 0.0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of 68 Ga-NOTA2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d. Conclusion:A new anti-HER2 PET tracer, 68 Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. 68 Ga-NOTA2Rs15d is ready for first-in-human clinical trials.

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, 99m Tc-7C12 and 99m Tc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99m Tc-tricarbonyl ( 99m Tc(CO) 3 ) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFRnegative) xenografts were intravenously injected with 99m Tc-7C12 and 99m Tc-7D12 on separate days. Pinhole SPECT/ micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six 57 Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm 3 ) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99m Tc-7C12 or 99m Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptorspecific. Image analysis showed high and similar tumor uptake values for both 99m Tc-7C12 and 99m Tc-7D12 (4.55 6 0.24 %IA/ cm 3 and 4.62 6 0.36 %IA/cm 3 , respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 6 0.14 for 99m Tc-7C12 and 1.49 6 0.60 for 99m Tc-7D12). 99m Tc-7C12 showed significantly higher kidney uptake (63.48 6 2.36 vs. 56.25 6 2.46 %IA/cm 3 ) and lower liver uptake (2.55 6 0.26 vs. 4.88 6 0.86 %IA/cm 3 ) than did 99m Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-tobackground ratio; however, 99m Tc-7C12 showed higher tumor uptake (9.11 6 1.12 %IA/g) than did 99m Tc-7D12 (6.09 6 0.77 %IA/g). 99m Tc-7D12 demonstrated significantly higher blood activity than did 99m Tc-7C12, but both showed short plasma half-lives (,10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, 99m Tc-7C12 was selected for further studies. Epi dermal growth factor receptor (EGFR or ErbB1) is a member of a receptor tyrosine kinase family together with Her-2-neu/ErbB2, HER-3/ErbB3, and HER-4/ErbB4. EGFR is implicated in many cellular processes such as proliferation, differentiation, and survival (1). Several reports have shown that EGFR signaling is abnormal in many tumors of epithelial origin, such as cancer of the breast, head and neck, prostate, lung, and skin. Aberrant signaling of...

Introduction: The combination of a targeted biomolecule that specifically defines the target and a radionuclide that delivers a cytotoxic payload offers a specific way to destroy cancer cells. Targeted radionuclide therapy (TRNT) aims to deliver cytotoxic radiation to cancer cells and causes minimal toxicity to surrounding healthy tissues. Recent advances using α-particle radiation emphasizes their potential to generate radiation in a highly localized and toxic manner because of their high level of ionization and short range in tissue. Areas covered: We review the importance of targeted alpha therapy (TAT) and focus on nanobodies as potential beneficial vehicles. In recent years, nanobodies have been evaluated intensively as unique antigen-specific vehicles for molecular imaging and TRNT. Expert opinion: We expect that the efficient targeting capacity and fast clearance of nanobodies offer a high potential for TAT. More particularly, we argue that the nanobodies’ pharmacokinetic properties match perfectly with the interesting decay properties of the short-lived α-particle emitting radionuclides Astatine-211 and Bismuth-213 and offer an interesting treatment option particularly for micrometastatic cancer and residual disease.