Preclinical Evaluation of a Nanobody-based SPECT Radiotracer ^99mTc-AFN with Clinical Potential for Noninvasive Monitoring of Fibroblast Activation after Myocardial Infarction

Purpose To explore the feasibility of a new technetium 99m-labeled nanobody (^99mTc-antifibroblast activation protein nanobody [AFN]) with high affinity to fibroblast activation protein for noninvasive monitoring of fibroblast activation after myocardial infarction. Materials and Methods ^99mTc-AFN was prepared by site-specific labeling. Dynamic SPECT/CT imaging from 0.5 to 4 hours after injection of ^99mTc-AFN was performed in male C57/BL6 mice at 8 weeks of age with the ischemia-reperfusion (IR) injury on day 7 (<i>n</i> = 4). Furthermore, IR mice underwent ^99mTc-AFN SPECT/CT imaging on postoperative days 3, 7, 14, and 28 at 1 hour following injection. Ex vivo SPECT/CT imaging, blocking imaging, and biodistribution studies were performed on day 7 and compared with a ^99mTc-labeled fibroblast activation protein inhibitor (FAPI)-04-derived radiotracer (^99mTc-HFAPi). Preclinical evaluation of ^99mTc-AFN in a swine model of myocardial infarction was performed on day 7. Biodistributions and quantitative results were compared between groups using analysis of variance and <i>t</i> tests. Results The accumulation of ^99mTc-AFN at the infarcted region was most clearly visible at 1 hour following injection. ^99mTc-AFN uptake on SPECT/CT images in the infarcted myocardium was visible from day 3, and maximum uptake occurred on day 7. At 1 hour after injection, the ^99mTc-AFN and ^99mTc-HFAPi uptake ratios (means ± SDs) were 2.23 ± 0.82 versus 1.34 ± 0.21 (<i>P</i> = .05), respectively, for infarcted to noninfarcted regions and 1.08 ± 0.49 versus 0.89 ± 0.42 (<i>P</i> = .34) for infarcted region to blood, respectively. Immunohistochemical examinations confirmed that maximum myocardium fibrosis and FAP expression was observed on day 7. ^99mTc-AFN uptake in the infarcted myocardium was also clearly visualized with the swine model of myocardial infarction. Conclusion This study demonstrated the potential of ^99mTc-AFN for noninvasive monitoring of fibroblast activation after myocardial infarction, which may be a new option for clinical imaging. <b>Keywords:</b> Fibroblast Activation Protein, Nanobody, ^99mTc-AFN, ^99mTc-HFAPi, Myocardial Infarction <i>Supplemental material is available for this article.</i> © RSNA, 2025.