Synthesis, Preclinical Validation, Dosimetry, and Toxicity of ⁶⁸Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer

Abstract

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.

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma

“…Nanobodies have proven to be ideal probes for SPECT and PET imaging in rodents, with high targeting potential and fast blood clearance (12,13). The anti-HER2-Nanobody 2Rs15 d was selected and optimized as the lead compound for clinical translation (14,15). In this paper we report on the safety, biodistribution, dosimetry, and tumor-targeting potential of the 68 Ga-anti-HER2-Nanobody ( 68 Ga-HER2-Nanobody) in breast carcinoma patients.…”

Positron Emission Tomography Imaging with 18F-Labeled ZHER2:2891 Affibody for Detection of HER2 Expression and Pharmacodynamic Response to HER2-Modulating Therapies

“…This permits utilization of more widely available short-lived radioisotopes, such as 18 F and 68 Ga, minimizing the patient's dosimetry. In comparison with nanobodies, Affibodies excel through lower K D , higher k on , and slower k off rates (36). Regarding affinity, surface plasmon resonance experiments with isotopically unmodified GE-226 revealed high affinity binding to human and rhesus HER2-ECD-Fc comparable to the binding of parent Z HER2:2891 Affibody to human HER2-ECD-Fc (76 pmol/L; ref.…”

Imaging Diagnostic and Therapeutic Targets: Human Epidermal Growth Factor Receptor 2

“…HER2 imaging has also been investigated with even smaller probes, namely Nanobodies (23,24). Many anti-HER2 Nanobodies for the noninvasive imaging of HER2 expression have been proposed (23).…”

“…Many anti-HER2 Nanobodies for the noninvasive imaging of HER2 expression have been proposed (23). Studies with Nanobodies radiolabeled with 99m Tc (2Rs15dHis6) in mice bearing HER2-positive tumors revealed fast blood clearance, low accumulation in nontarget organs (except the kidneys, which are responsible for tracer clearance), and high concomitant tumor-to-blood and tumor-to-muscle ratios at 1 h after injection (24). PET imaging of HER2 expression with 68 Ga-radiolabeled Nanobodies is currently being evaluated by the same group of researchers ( Fig.…”