Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET

The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. Immuno-PET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor-targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; similar to 13 kDa) after F-18 labeling by 2 methods. Methods: The 5F7 Nanobody was labeled with F-18 using the novel residualizing label N-succinimidyl 3-((4-(4-F-18-fluorobutyl)-1 H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate (F-18-SFBTMGMB; F-18-RL-I) and also via the most commonly used F-18 protein-labeling prosthetic agent N-succinimidyl 3-F-18-fluorobenzoate (F-18-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-I-125-iodobenzoate (I-125-SGMIB). Paired label (F-18/I-125) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Small-animal PET/CT imaging of 5F7 Nanobody labeled using F-18-RL-I also was performed. Results: Internalization assays indicated that intracellularly retained radioactivity for F-18-RL-I-5F7 was similar to that for coincubated I-125-SGMIB-5F7, whereas that for F-18-SFB-5F7 was lower than coincubated I-125-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of F-18-RL-I-5F7 was 28.97 3.88 percentage injected dose per gram of tissue (%ID/g) at 1 h and 36.28 +/- 14.10 %ID/g at 2 h, reduced by more than 90% on blocking with trastuzumab, indicating HER2 specificity of uptake, and was also 26%-28% higher (P < 0.05) than that of F-18-SFB5F7. At 2 h, the tumor-to-blood ratio for F-18-RL-I-5F7 (47.4 +/- 13.1) was significantly higher (P < 0.05) than for F-18-SFB-5F7 (25.4 +/- 10.3); however, kidney uptake was 28-36-fold higher for F-18-RL-I-5F7. Conclusion: F-18-RL-I-5F7 is a promising tracer for evaluating HER2 status by immuno-PET; however, in settings in which renal background is problematic, strategies for reducing its kidney uptake may be needed.