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

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 ß-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.

Two-photon excited fluorescence (TPEF) may be useful to identify macrophage subsets based on their metabolic activity and cellular responses in atherosclerotic plaques.

BACKGROUND AND AIMS: Atherosclerosis is characterized by the formation of lipid plaques within the arterial wall. In such plaques, the massive and continuous recruitment of circulating monocyte-derived macrophages induces inflammation, leading to plaque destabilization and rupture. Plaque vulnerability is linked to the presence of (i) a large lipid core that contains necrotic, "foamy" macrophages (FMs), (ii) a thin fibrous cap that cannot limit the prothrombotic lipid core, and potentially (iii) an imbalance between inflammatory and immunoregulatory macrophages. These opposite macrophage functions rely on the use of different energy pathways (glycolysis and oxidative phosphorylation, respectively) that may lead to different levels of the auto-fluorescent cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We hypothesized that high-resolution two-photon excited autofluorescence (TPEF) imaging of these cofactors may be used to monitor the metabolic activity and cellular responses of macrophages in atherosclerotic plaques. METHODS: Different models of human FMs were generated by exposure to acetylated or oxidized low-density lipoproteins (LDL), with/without human carotid extract (CE). Their phenotype and optical properties were compared with those of extremely polarized macrophages, inflammatory M1 (MLPS+IFNγ) and immunoregulatory M2 (MIL4+IL13). RESULTS: These FM models displayed an intermediate phenotype with low levels of M1 and M2 "specific" markers. Moreover, the NADH and FAD autofluorescence profiles of FMoxLDL ± CE cells were significantly distinct from those of M1 and M2 macrophages. CONCLUSIONS: TPEF imaging may be useful to follow the metabolic activity and cellular responses of the different macrophage subtypes present in atherosclerotic plaques in order to detect vulnerable areas.

Recent Advances in the Molecular Imaging of Atherosclerosis.

Atherosclerosis is the major underlying cause of cardiovascular diseases, the prevalence of which is continuously increasing, thus currently standing as the leading global cause of death. This pathology gradually develops over the course of 50 or more years throughout the life of an individual under the influence of a vast number of factors, both environmental and pathophysiological. This wealth of factors has elicited much research into molecular imaging, with purely diagnostic purposes or with the hope of engineering an efficient theranostic tool. To these ends, diverse nanomaterials with desirable, tunable properties have been explored by different teams, as described in this review.

Multimodal molecular imaging of atherosclerosis: Nanoparticles functionalized with scFv fragments of an anti-αIIbß3 antibody.

Due to the wealth of actors involved in the development of atherosclerosis, molecular imaging based on the targeting of specific markers would substantiate the diagnosis of life-threatening atheroma plaques. To this end, TEG4 antibody is a promising candidate targeting the activated platelets (integrin αIIbß3) highly represented within the plaque. In this study, scFv antibody fragments were used to functionalize multimodal imaging nanoparticles. This grafting was performed in a regio-selective way to preserve TEG4 activity and the avidity of the nanoparticles was studied with respect to the number of grafted antibodies. Subsequently, taking advantage of the nanoparticle bimodality, both near infrared fluorescence and magnetic resonance imaging of the atheroma plaque were performed in the ApoE-/- mouse model. Here we describe the design of the targeted nanoparticles, and a quantification method for their detection in mice, both ex vivo and in vivo, highlighting their value as a potential diagnosis agent.

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

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.

Data on atherosclerosis specific antibody conjugation to nanoemulsions.

This article present data related to the publication entitled "Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging" (Prévot et al., 2017) [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs) using a heterobifunctional linker (DSPE-PEG-maleimide). Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging.

PURPOSE: For early atherosclerosis imaging, magnetic oil-in-water nanoemulsion (NE) decorated with atheroma specific monoclonal antibody was designed for Magnetic Particle Imaging (MPI) and Magnetic Resonance Imaging (MRI). MPI is an emerging technique based on direct mapping of superparamagnetic nanoparticles which may advantageously complement MRI. METHODS: NE oily droplets were loaded with superparamagnetic iron oxide nanoparticles of 7, 11 and 18nm and biofunctionalized with atheroma specific scFv-Fc TEG4-2C antibody. RESULTS: Inclusion of nanoparticles inside NE did not change the hydrodynamic diameter of the oil droplets, close to 180nm, nor the polydispersity. The droplets were negatively charged (ζ=-30mV). In vitro MPI signal was assessed by Magnetic Particle Spectroscopy (MPS). NE displayed MRI and MPS signals confirming its potential as new contrast agent. NE MPS signal increase with NPs size close to the gold standard (Resovist). In MRI, NE displayed R2* transversal relaxivity of 45.45, 96.04 and 218.81mM-1s-1 for 7, 11 and 18nm respectively. NE selectively bind atheroma plaque both in vitro and ex vivo in animal models of atherosclerosis. CONCLUSION: Magnetic NE showed reasonable MRI/MPS signals and a significant labelling of the atheroma plaque. These preliminary results support that NE platform could selectively image atherosclerosis.

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting.

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbß3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbß3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.

Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an In Vivo Selected Phage-Display Combinatorial Library.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both in vivo and in vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800 bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an in vivo selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the in vivo selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the in vivo panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767 bp, 53 of them covering the whole insert of 977 bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939 bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

Nanoparticles functionalised with an anti-platelet human antibody for in vivo detection of atherosclerotic plaque by magnetic resonance imaging.

Atherosclerosis is an inflammatory disease associated with the formation of atheroma plaques likely to rupture in which platelets are involved both in atherogenesis and atherothrombosis. The rupture is linked to the molecular composition of vulnerable plaques, causing acute cardiovascular events. In this study we propose an original targeted contrast agent for molecular imaging of atherosclerosis. Versatile USPIO (VUSPIO) nanoparticles, enhancing contrast in MR imaging, were functionalised with a recombinant human IgG4 antibody, rIgG4 TEG4, targeting human activated platelets. The maintenance of immunoreactivity of the targeted VUSPIO against platelets was confirmed in vitro by flow cytometry, transmission electronic and optical microscopy. In the atherosclerotic ApoE(-/-) mouse model, high-resolution ex vivo MRI demonstrated the selective binding of TEG4-VUSPIO on atheroma plaques. It is noteworthy that the rationale for targeting platelets within atherosclerotic lesions is highlighted by our targeted contrast agent using a human anti-αIIbß3 antibody as a targeting moiety. FROM THE CLINICAL EDITOR: Current clinical assessment of atherosclerotic plagues is suboptimal. The authors in the article designed functionalized superparamagnetic iron oxide nanoparticles with TEG4, a recombinant human antibody, to target activated platelets. By using MRI, these nanoparticles can be utilized to study the process of atheroma pathogenesis.

By-passing large screening experiments using sequencing as a tool to identify scFv fragments targeting atherosclerotic lesions in a novel in vivo phage display selection.

Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. To interrogate the molecular components involved in this process, single-chain variable fragments (scFvs) from a semi-synthetic human antibody library were selected on the lesions induced in a rabbit model of atherosclerosis after two rounds of in vivo phage display. Homing Phage-scFvs were isolated from (1) the injured endothelium, (2) the underlying lesional tissue and (3) the cells within the intima. Clones selected on the basis of their redundancy or the presence of key amino acids, as determined by comparing the distribution between the native and the selected libraries, were produced in soluble form, and seven scFvs were shown to specifically target the endothelial cell surface and inflamed intima-related regions of rabbit tissue sections by immunohistology approaches. The staining patterns differed depending on the scFv compartment of origin. This study demonstrates that large-scale scFv binding assays can be replaced by a sequence-based selection of best clones, paving the way for easier use of antibody libraries in in vivo biopanning experiments. Future investigations will be aimed at characterizing the scFv/target couples by mass spectrometry to set the stage for more accurate diagnostic of atherosclerosis and development of therapeutic strategies.

Development of a platform of antibody-presenting liposomes.

Antibody-presenting liposomes present high interest as drug delivery systems. The association of antibodies to liposomes is usually realized by covalent coupling of IgGs or their antigen-binding fragments to lipid polar head groups by means of hetero-bifunctional crosslinkers. We present here an original platform of IgG-presenting liposomes which is based on a fusion protein between Annexin-A5 (Anx5) and the IgG-binding ZZ repeat derived from Staphylococcus aureus protein A. The Anx5ZZ fusion protein acts as a bi-functional adaptor that anchors IgGs to liposomes in a non covalent and highly versatile manner. The interactions between IgGs, Anx5ZZ and liposomes were characterized by PAGE, dynamic light scattering and fluorescence quenching assays, establishing that binding of Anx5ZZ to IgGs and of Anx5ZZ-IgG complexes to liposomes is complete with stoichiometric amounts of each species. We found that the sequence of assembly is important and that Anx5ZZ-IgG complexes need to be formed first in solution and then adsorbed to liposomes in order to avoid aggregation. The targeting capacity of Anx5ZZ-IgG-functionalized liposomes was demonstrated by electron microscopy on an ex vivo model system of atherosclerotic plaques. This study shows that the Anx5ZZ adaptor constitutes an efficient platform for functionalizing liposomes with IgGs. This platform may present potential applications in molecular imaging and drug delivery.

Discovery and characterization of an Arabidopsis thaliana N-acylphosphatidylethanolamine synthase.

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D beta/gamma isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. beta-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling.

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.

Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis.

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

Production of a human monoclonal IgM directed against human cardiac myosin in a hollow-fiber bioreactor for membrane anion exchange chromatography one-step purification.

Purification of human IgM monoclonal antibodies (MAbs) has proved to be difficult. Since IgM Mabs tend to bind strongly to a variety of resin support surfaces, the number of chromatographic steps used in the purification of these biomolecules should be minimized. Here we describe procedures developed for the optimal production and purification of the human monoclonal IgM B7, which specifically binds to the myosin heavy chain of human ventricular myocardium. This property makes this antibody potentially useful for the diagnosis of myocardial necrosis. Several chromatographic techniques were evaluated (size exclusion, ion exchange, affinity chromatography). The best results were obtained with anion exchange membrane chromatography using Sartobind Q15 (98% purity, 30% recovery). IgM production was improved by the hollow fiber technology which permitted the use of serum-reduced medium and an increase in antibody concentration to an average production of 300-400 microg/ml, compared to 20 microg/ml in flask culture. Several flow-rates were also evaluated, the optimal being 20 ml/minute for 30% of recovery. Importantly, the purified IgM molecule was able to bind to human myosin in ELISA and Western-blotting, thus allowing the IgM to be kept intact for further radiolabeling.

Human IgG monoclonal anti-alpha(IIb)beta(3)-binding fragments derived from immunized donors using phage display.

Previous studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have relied on serum analysis and have shown the frequent development of Abs directed against the alpha(IIb)beta(3) integrin. However, little is known about the molecular diversity of the humoral immune response to alpha(IIb)beta(3) due to the paucity of mAbs issuing from these pathologies. We have isolated human IgG anti-alpha(IIb)beta(3) binding fragments using combinatorial libraries of single-chain IgG created from the B cells of a GT and an AITP patient, both with serum Abs. Ab screening was performed using activated platelets or activated alpha(IIb)beta(3)-expressing Chinese hamster ovary cells. Sequencing of selected phage Abs showed that a broad selection of genes from virtually all V gene families had been used, indicating the diversity of the immune response. About one-half of the V(H) and V(L) segments of our IgG anti-alpha(IIb)beta(3) fragments displayed extensive hypermutations in the complementarity-determining region, supporting the idea that an Ag-driven immune response was occurring in both patients. The H chain complementarity-determining region 3 analysis of phage Abs revealed motifs other than the well-known RGD and KQAGDV integrin-binding sequences. To our knowledge, our study is the first to illustrate multiple human IgG anti-alpha(IIb)beta(3) reactivities and structural variations linked to the anti-platelet human immune response. Human alpha(IIb)beta(3) Abs preferentially directed against the activated form of the integrin were further characterized because platelet alpha(IIb)beta(3) inhibitors are potential therapeutic reagents for treating acute coronary syndromes. Currently available alpha(IIb)beta(3) antagonists do not specifically recognize the activated form of the integrin.