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In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis
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Edité par CCSD ; Wiley-Blackwell -
International audience. BACKGROUND: Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages,and lipid-laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed toidentify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment.METHODS AND RESULTS: Human scFv (single-chain fragment variable) selected by in vivo phage display in a rabbit model ofatherosclerosis was reformatted as scFv fused to the scFv-Fc (single-chain fragment variable fused to the crystallizable frag-ment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aortasections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins fromhuman endarterectomies was co-immunoprecipitated with the selected scFv-Fc followed by mass spectrometry for targetidentification. Near-infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647– labeledscFv-Fc-2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano-objects for theranostic applications. One scFv-Fc clone (P3) displayed the highest cross-reactivity against atheroscleroticlesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identifiedgalectin-3, a β-galactoside-binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercialanti-galectin-3 antibody confirmed this specificity. P3 scFv-Fc-2c specifically targeted atherosclerotic plaques in the Apoe −/−mouse model.CONCLUSIONS: These results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclero-sis and other inflammatory pathologies involving macrophages. Recently, galectin-3 was proposed as a high-value biomarkerfor the assessment of coronary and carotid atherosclerosis.
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