A Novel P2X7-targeted Nanobody Radiotracer 99mTc-1c81 for SPECT Imaging of Atherosclerotic Plaques

Introduction: The rupture of vulnerable atherosclerotic plaque (VAP) is the most common cause of coronary thrombotic events and sudden cardiac death. Early diagnose and timely intervention of VAP are urgent in clinic. P2X7 receptor, which is expressed mainly in pro-inflammatory macrophages, has been considered as a potential new target for detecting and treating VAP. Nanobodies are the smallest antibody fragment with high antigen binding ability and specificity, which are well-suited for radionuclide imaging. The present study aimed to design a novel P2X7-targeted nanobody based SPECT probe for identification of atherosclerotic plaque (AP).

Methods: ^99mTc-HYNIC-GGGGC (GGGGC = NH2-Gly-Gly-Gly-Gly-Cys-COOH, HYNIC = 6-hydrazinonicotinyl) was prepared firstly and then site-specifically connected to anti-P2X7 nanobody 1c81 reported previously by transpeptidation of Sortase A. The affinity and specificity of ^99mTc-1c81 were evaluated by saturation binding experiment and binding assay in RAW264.7 cells, respectively. The biodistribution study of ^99mTc-1c81 was performed in C57 mice at 0.5 h, 1 h, 2 h, and 4 h post injection (p.i.), and SPECT/CT imaging was underwent in ApoE^-/- (20 weeks on high-fat diet) and C57 mice (20 weeks on normal diet) at 2 h p.i., respectively. Target to background ratio (TBR) values in AP were calculated as the ratio of the mean count per cubic centimeter in the aortic arch to the mean count per cubic centimeter in the cardiac blood-pool. The specific binding of ^99mTc-1c81 in the AP was further confirmed by autoradiography and Oil Red O staining. In order to validate and correlate with imaging findings, the lipid distribution and the expression of P2X7 receptor in aortic valve sections were evaluated by Oil Red O and immunohistochemistry stainings, respectively.

Results: The labelling yield of ^99mTc-HYNIC-GGGGC determined by RP-HPLC was > 95% after 10 minutes of reaction at 100 ℃. The enzymatic reaction between the 1c81 and ^99mTc-HYNIC-GGGGC was completed at 37 ℃ in 20 minutes. After purification by NAP-5 column, ^99mTc-1c81 was synthesized in 50% radiochemical yield with radiochemical purity of > 99% and molar activity of > 11 MBq/nmol (n = 3). In the saturation binding experiment, ^99mTc-1c81 exhibited high affinity for P2X7 receptor, with the binding equilibrium dissociation constant of 6.375 nM. The binding assay revealed that significant higher cellular uptake of ^99mTc-1c81 in binding group than blocking group (incubated with 1000-fold molar 1c81) in P2X7 high expression RAW264.7 cells, which indicated that the specific binding of ^99mTc-1c81 to the P2X7 receptor (% added dose per 10⁶ cells: 2.34 ± 0.11 vs 0.08 ± 0.01, n = 4, P < 0.0001). Biodistribution results revealed that ^99mTc-1c81 was rapidly cleared from the blood and normal organs except for the kidneys. SPECT imaging of ^99mTc-1c81 in the aortic arch demonstrated a clear visualization of AP and the TBR of ApoE^-/- group was significantly higher than that of C57 group at 2 h p.i (TBR: 4.49 ± 1.88 vs 0.96 ± 0.64, n = 4, P = 0.012), indicating that ^99mTc-1c81 could efficiently detect AP in the aortic arch. The autoradiography results showed that the radioactive uptake of ^99mTc-1c81 was colocalized well with the AP, indicating the specificity of ^99mTc-1c81 in vivo. Additionally, high expression of P2X7 receptor in the AP of aortic valve sections was confirmed by immunohistochemical staining in vitro.

Conclusions: ^99mTc-1c81 exhibited specific binding to P2X7 receptor in AP in vivo. It may serve as a novel P2X7-targeted SPECT tracer to evaluate the vulnerability of AP.

Acknowledgements: This work was supported by the Beijing Hospitals Authority Clinical Medicine Development of special funding support (ZYLX202110), National Natural Science Foundation of China (82171994), Beijing Municipal Natural Science Foundation (7232040), Advanced Research Foundation of Capital Medical University (PYZ22127), Science and Technology Development Fund of Beijing Anzhen Hospital (AZYZR202202).

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