In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

Abstract

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 to identify 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 of atherosclerosis was reformatted as scFv fused to the scFv‐Fc (single‐chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co‐immunoprecipitated with the selected scFv‐Fc followed by mass spectrometry for target identification. Near‐infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647–labeled scFv‐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 atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin‐3, a β‐galactoside‐binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti‐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 atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin‐3 was proposed as a high‐value biomarker for the assessment of coronary and carotid atherosclerosis.

Defining atherosclerotic plaque biology by mass spectrometry-based omics approaches

“…69 Immunoprecipitation followed by GeLCMS on human endarterectomy samples showed the utilization of galectin-3 for developing improved imaging-based diagnostics and biotherapeutics. 70 The proteomic and peptidome analysis of atherosclerotic lesions from carotid endarterectomy by Jin H. et al stratified the plaques with different phenotypes like stable, vulnerable and hemorrhage with high predictive power. Also, it revealed the association of a transcription factor, serum response factor (SRF), with the plaque phase transition.…”

In vivo Phage Display: A promising selection strategy for the improvement of antibody targeting and drug delivery properties

“…The identification of the promising P3 scFv antibody antigen allowed to propose galectin-3 as an ideal molecular biomarker that can be targeted for diagnostic purposes. Curiously, galectin-3 has been recently proposed as a high-value biomarker for the assessment of coronary and carotid atherosclerosis ( Hemadou et al, 2021 ). Notably, in vivo phage display has also been used in diabetes research field.…”