Development of CD163 targeted radiotracer for imaging M2 anti-inflammatory macrophages

Introduction: Dynamic variation of macrophage subsets during promotion and resolution of inflammation is of great interest since they play diverse roles in the acute response to infection and tissue injury, tissue repair, and cancer development. Typically, macrophages are defined as M1 (classically activated pro-inflammatory macrophages) and M2 (alternatively activated tissue-resident anti-inflammatory macrophages) subsets. Our group has developed ⁶⁴Cu-/⁶⁸Ga-DOTA-ECL1i that can detect CCR2, a representative biomarker for M1 macrophages. To monitor temporal-spatial distribution of M1 and M2 macrophages in inflammatory diseases and malignancies, herein, our goal is to develop novel CD163 targeted PET radiotracers that can detect M2 macrophages.

Methods: We performed phage display screening of CD163 binding peptides using the Ph.D.-12^TM library and CD163 protein. We identified a leading sequence named ICT-01 and conjugated a NODAGA chelator to N-terminus by amidation reaction. The resulting NODAGA-ICT-01 precursor was radiolabeled with ⁶⁴Cu or ⁶⁸Ga. The in vitro cell binding assay of ⁶⁴Cu-NODAGA-ICT-01 was performed in CD163 overexpressing U87 cells. In vivo pharmacokinetics of ⁶⁴Cu-NODAGA-ICT-01 was performed in wild type C57BL/6 mice at 1 h, 2 h, and 4 h post tail vein injection. ⁶⁴Cu-/⁶⁸Ga-NODAGA-ICT-01 PET/CT imaging of CD163 was assessed in an ApoE^-/- mouse atherosclerosis model and a reperfused myocardial infarction mouse model. The competitive blocking study was performed to confirm specificity. In an ischemia reperfusion injury induced myocardial infarction mouse model, CCR2 and CD163 PET imaging was conducted with ⁶⁸Ga-DOTA-ECL1i and ⁶⁸Ga-NODAGA-ICT-01, respectively, over the course of two consecutive days for comparison.

Results: ⁶⁴Cu-NODAGA-ICT-01 demonstrated high binding affinity to CD163 with an IC₅₀ of 26.8 ± 20.7 nM (n=3). ⁶⁴Cu-NODAGA-ICT-01 showed effective renal clearance and low retention in most organs. The blood pool organs (blood, heart, lung) had less than 0.5 %ID/g retention at 1 h post injection while the liver uptake was less than 2 %ID/g at all time points. Interestingly, gallbladder showed high retention of ⁶⁴Cu-NODAGA-ICT-01 (31.8 ± 8.1 %ID/g) at 1 h, followed by rapid decrease to less than 4 %ID/g at 4 h, which still ensured acceptable dosimetry for potential clinical translation based on the preliminary estimation. Consistent with the rapid blood clearance determined in the biodistribution study, low tracer uptake was observed at the aortic arch in WT mice. In contrast to the low accumulation of ¹⁸F-FDG in ApoE^-/- mice after 30 weeks on high fat diet (HFD), a strong signal (1.59 ± 0.20 %ID/g) from CD163 PET imaging was determined at the aortic arch in ApoE^-/- mice after only 10 weeks on HFD and almost doubled (2.89 ± 0.49 %ID/g) at 40 weeks HFD. These results are consistent with the increased M2 resident macrophages in atherosclerosis. The competitive blocking study revealed approximately 3-fold decrease of tracer uptake, suggesting the targeting specificity. In a reperfused myocardial infarction mouse model, day 4 CCR2 PET revealed intense tracer uptake (1.42 ± 0.11 %ID/g) at the infarct zone, which gradually decreased through the remodeling process. On the other hand, CD163 PET signals were observed at the remote area and gradually increased from day 5 (1.54 ± 0.16 %ID/g) to day 13 (2.92 ± 0.35 %ID/g) and then remained stable until day 25.

Conclusions: We have identified and synthesized a candidate tracer ⁶⁴Cu-/⁶⁸Ga-NODAGA-ICT-01 for CD163 PET imaging. Initial evaluations demonstrate the stability of the radiotracer in mouse serum and in vitro cell binding studies show high binding affinity to CD163. Biodistribution showed fast renal clearance and low retention in major organs except the gallbladder in WT mice. PET imaging in both an ApoE^-/- atherosclerotic mouse model and a reperfused myocardial infarction mouse model demonstrated the potential of ⁶⁴Cu-/⁶⁸Ga-NODAGA-ICT-01 for further evaluation in inflammatory animal models.