An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis

Abstract

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis.

Challenges in the development of nanoparticle‐based imaging agents: Characterization and biology

“…In an in vivo phage display screen, animals are euthanized and perfused after the desired time point (~5–10 min for identifying phage specific for endothelium, or ~4–6 hr for identifying phage specific for cells within a desired tissue) following i.v. administration and phage is recovered from the tissue or a section of tissue by homogenizing and amplifying in Escherichia coli (Siva Sai Krishna Dasa et al, 2015; Deramchia et al, 2012; Gutknecht et al, 2017; Hemadou et al, 2018). Target specific phage is amplified in bacteria, and by doing additional rounds of selection, high affinity phage clones can be obtained.…”

Recent Advances in the Molecular Imaging of Atherosclerosis

“…Identification of relevant biomarkers is the stepping stone for an efficient molecular imaging using different modalities. Recent studies highlight the capacity of combining in vivo phage display (using a human combinatorial antibody library) and high-throughput biological screening on atheroma extracts 187 and/or in silico tracking of the most frequently used antibody sequences 188 to select potent and valuable antibody candidates. Further characterization of the targeted molecules by proteomic approaches would give new insights into in vivo targetable, upregulated biomarkers of atherosclerosis.…”

A Nano-Emulsion Platform Functionalized with a Fully Human scFv-Fc Antibody for Atheroma Targeting: Towards a Theranostic Approach to Atherosclerosis

“…P3 specifically targets galectin-3, a protein that has been highlighted in recent studies as a new atherosclerosis biomarker [ 55 ]. P3 was discovered by in vivo phage-display selection in an animal model of atherosclerosis using a human scFv library [ 56 , 57 ]. Its ability to target galectin-3 within atherosclerotic lesions has been demonstrated in vitro and ex vivo (patent WO2019068863A1).…”

“…IHC experiments were performed as previously described [ 31 ]. Briefly, after deparaffinization, heat-induced epitope retrieval and non-specific interaction blocking, as described in [ 56 ], aorta sections were incubated with NE-P3 ((#5), 53 µg/mL of P3 antibody, 158 mg/L of Fe), unconjugated NE–PEG 3400 –maleimide ((#3), 158 mg/L of Fe), ScFv-Fc-2Cys P3 HuAb (53 µg/L) (positive control), or the diluent alone (negative control) overnight. This was followed by incubation with the secondary HRP-conjugated goat anti-human antibody (Fcγ-specific; 1:1000 ( v / v ) (Jackson ImmunoResearch; West Grove, PA, USA), and antibody binding was revealed using the Dako Liquid DAB+ Substrate Chromogen System (Agilent Technologies Inc, Santa Clara, CA, USA).…”