CD163 Targeted Peptide Screened by Phage Display as PET Tracer Imaging Resident Macrophages in Atherosclerosis

Introduction: Tissue resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The non-invasive detection of their presence and dynamic variation will be important to understand their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163+ macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans.

Methods: Phage display screening of CD163 binding peptide was performed with a Ph D-12^TM phage display peptide library. The initially identified peptide sequences were further screened with phage and peptide ELISA with CD163 protein. A lead peptide was selected for conjugation with chelator for radiolabeling (⁶⁴Cu-ICT-01), which was confirmed by mass spectrometry. CD163 overexpressing U87 cells were used for cell binding to determine IC₅₀ values. Biodistribution studies were performed in wild type C57BL/6 mice at 1h, 2h, and 4h post tail vein injection. The sensitivity and specificity of ⁶⁴Cu-ICT-01 imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining and flow cytometry were performed to characterize the expression of CD163 on tissue resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of ⁶⁴Cu-ICT-01.

Results: Eighteen sequences of 50 colonies were identified after 3 rounds of bio-screening with Ph D-12^TM phage display peptide library. Five peptides were selected for tracer development after phage and peptide ELISA. An identified lead peptide was radiolabeled after conjugation with NODAGA for next phase screening. The radiolabeling specific activity of ⁶⁴Cu-ICT-01 was calculated as 7.41×10¹⁰MBq·mol^-1. The IC₅₀ of ⁶⁴Cu-ICT-01 was determined to be 14.4±1.0 nM in U87 cells. Biodistribution in wild type C57BL/6 mice showed low blood retention, effective renal clearance, and rapidly decreased gallbladder accumulation. In an ApoE^-/- mouse model, ⁶⁴Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability to track the progression of atherosclerotic lesions, which was further confirmed in Ldlr^-/- and proprotein convertase subtilisin/kexin type 9 (PCSK9) mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry confirmed the specific expression of CD163 on tissue resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions and ex vivo autoradiography revealed specific binding of ⁶⁴Cu-ICT-01 to human CD163.

Conclusions: This study demonstrated the successful development of a novel CD163 radiotracer binding to CD163+ macrophages by using phage display peptide screening. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of ⁶⁴Cu-ICT-01 imaging CD163+ macrophages warrant further investigation in translational settings.