2024-2021

1-Phenotypic, Metabolic, and Functional Characterization of Experimental Models of Foamy Macrophages: Toward Therapeutic Research in Atherosclerosis.

Henni Mansour AS, Ragues M, Brevier J, Borowczyk C, Grevelinger J, Laroche-Traineau J, Garaude J, Marais S, Jacobin-Valat MJ, Gerbaud E, Clofent-Sanchez G, Ottones F. Int J Mol Sci. 2024 Sep 21;25(18):10146. doi: 10.3390/ijms251810146. PMID: 39337629

Different types of macrophages (Mφ) are involved in atherogenesis, including inflammatory Mφ and foamy Mφ (FM). Our previous study demonstrated that two-photon excited fluorescence (TPEF) imaging of NADH and FAD autofluorescence (AF) could distinguish experimental models that mimic the different atherosclerotic Mφ types. The present study assessed whether optical differences correlated with phenotypic and functional differences, potentially guiding diagnostic and therapeutic strategies. Phenotypic differences were investigated using three-dimensional principal component analysis and multi-color flow cytometry. Functional analyses focused on cytokine production, metabolic profiles, and cellular oxidative stress, in LDL dose-dependent assays, to understand the origin of AF in the FAD spectrum and assess FM ability to transition toward an immunoregulatory phenotype and function. Phenotypic studies revealed that FM models generated with acetylated LDL (Mac) were closer to immunoregulatory Mφ, while those generated with oxidized LDL (Mox) more closely resembled inflammatory Mφ. The metabolic analysis confirmed that inflammatory Mφ primarily used glycolysis, while immunoregulatory Mφ mainly depended on mitochondrial respiration. FM models employed both pathways; however, FM models generated with high doses of modified LDL showed reduced mitochondrial respiration, particularly Mox FM. Thus, the high AF in the FAD spectrum in Mox was not linked to increased mitochondrial respiration, but correlated with the dose of oxidized LDL, leading to increased production of reactive oxygen species (ROS) and lysosomal ceroid accumulation. High FAD-like AF, ROS, and ceroid accumulation were reduced by incubation with α-tocopherol. The cytokine profiles supported the phenotypic analysis, indicating that Mox FM exhibited greater inflammatory activity than Mac FM, although both could be redirected toward immunoregulatory functions, albeit to different degrees. In conclusion, in the context of immunoregulatory therapies for atherosclerosis, it is crucial to consider FM, given their prevalence in plaques and our results, as potential targets, regardless of their inflammatory status, alongside non-foamy inflammatory Mφ.

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

Hemadou A, Fontayne A, Laroche-Traineau J, Ottones F, Mondon P, Claverol S, Ducasse É, Sanchez S, Mohamad S, Lorenzato C, Duonor-Cerutti M, Clofent-Sanchez G, Jacobin-Valat MJ. J Am Heart Assoc. 2021 Oct 5;10(19):e016287. doi: 10.1161/JAHA.120.016287. Epub 2021 Sep 25. PMID: 34569248

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 beta-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.

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

Bonnet S, Prévot G, Mornet S, Jacobin-Valat MJ, Mousli Y, Hemadou A, Duttine M, Trotier A, Sanchez S, Duonor-Cérutti M, Crauste-Manciet S, Clofent-Sanchez G. Int J Mol Sci. 2021 May 14;22(10):5188. doi: 10.3390/ijms22105188. PMID: 34068875

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a « theranostic approach » that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe(-/-) mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.

2020-2018

1-Recent Advances in the Molecular Imaging of Atherosclerosis.

Larivière M, Bonnet S, Lorenzato C, Laroche-Traineau J, Ottonès F, Jacobin-Valat MJ, Clofent-Sanchez G. Semin Thromb Hemost. 2020 Jul;46(5):563-586. doi: 10.1055/s-0039-1701019. Epub 2020 Jun 30. PMID: 32604420 Review.

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.

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

Borowczyk C, Laroche-Traineau J, Brevier J, Jacobin-Valat MJ, Marais S, Gerbaud E, Clofent-Sanchez G, Ottones F. Atherosclerosis. 2020 Sep;309:47-55. doi: 10.1016/j.atherosclerosis.2020.07.017. Epub 2020 Jul 30. PMID: 32871394

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+IFNgamma) 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.

3-Development of anti-chloro 192 tyrosine HDL apoA-I antibodies for the immunodiagnosis of cardiovascular diseases.

Lokeshwaran K, Hemadou A, Jayaprakash NS, Prasanna RR, Jacobin-Valat MJ, Dieryck W, Joucla G, Vijayalakshmi MA, Clofent-Sanchez G, Santarelli X, Venkataraman K. J Immunol Methods. 2019 Nov;474:112637. doi: 10.1016/j.jim.2019.112637. Epub 2019 Aug 3. PMID: 31386835

High density lipoproteins (HDL) are considered cardio protective. Apolipoprotein A-I (apoA-I), a major component of HDL helps in reverse cholesterol transport, whose function is greatly affected during atherosclerosis due to oxidation by myeloperoxidase. Amino acid tyrosine residue of apoA-I at position 192 and 166 are sensitive to oxidation by myeloperoxidase resulting in the generation of chlorinated and nitrated apoA-I and they are believed to be present in atherosclerotic plaques and in circulation. These oxidized apoA-I have been suggested as potential indicator(s) of CVD risks in humans. To detect the levels of oxidized apoA-I there is a need for developing monoclonal antibodies (mAbs) with high specificity and sensitivity that could be utilized routinely in clinical immune based assays for blood plasma or for in vivo imaging. In this study, chemically chlorinated apoA-I (chlorinated (192)tyrosine- apoA-I) and a short synthetic peptide, containing the corresponding chlorinated tyrosine residue, conjugated to keyhole limpet hemocyanin (KLH) carrier protein were used for immunization. Stable hybridoma clones F7D5 and G11E3 were found to be highly sensitive and reactive towards chlorinated (192)tyrosine- apoA-I. Interestingly, these mAbs also displayed positive reaction with atherosclerotic plaques obtained from mouse and human biopsies. In vitro or in vivo diagnostic tests could be developed either by detecting oxidized apoA-I in human plasma or by directly imaging atheroma plaques as both mAbs were shown to stain human atheroma. The anti-chlorinated (192)tyrosine- apoA-I mAbs described in this study may have a high diagnostic potential in predicting CVD risks.

4-Multimodal molecular imaging of atherosclerosis: Nanoparticles functionalized with scFv fragments of an anti-αIIbβ3 antibody.

Larivière M, Lorenzato CS, Adumeau L, Bonnet S, Hémadou A, Jacobin-Valat MJ, Noubhani A, Santarelli X, Minder L, Di Primo C, Sanchez S, Mornet S, Laroche-Traineau J, Clofent-Sanchez G. Nanomedicine. 2019 Nov;22:102082. doi: 10.1016/j.nano.2019.102082. Epub 2019 Aug 9. PMID: 31404651

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 alphaIIbbeta3) 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.

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

Hemadou A, Laroche-Traineau J, Antoine S, Mondon P, Fontayne A, Le Priol Y, Claverol S, Sanchez S, Cerutti M, Ottones F, Clofent-Sanchez G, Jacobin-Valat MJ. Sci Rep. 2018 Oct 9;8(1):15016. doi: 10.1038/s41598-018-33382-2. PMID: 30302027

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.

2017-2015

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

Prévot G, Kauss T, Lorenzato C, Gaubert A, Larivière M, Baillet J, Laroche-Traineau J, Jacobin-Valat MJ, Adumeau L, Mornet S, Barthélémy P, Duonor-Cérutti M, Clofent-Sanchez G, Crauste-Manciet S. Int J Pharm. 2017 Nov 5;532(2):669-676. doi: 10.1016/j.ijpharm.2017.09.010. Epub 2017 Sep 9. PMID: 28899764

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 (zeta=-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(-1)s(-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.

2-Data on atherosclerosis specific antibody conjugation to nanoemulsions.

Prévot G, Duonor-Cérutti M, Larivière M, Laroche-Traineau J, Jacobin-Valat MJ, Barthélémy P, Clofent-Sanchez G, Crauste-Manciet S. Data Brief. 2017 Oct 26;15:824-827. doi: 10.1016/j.dib.2017.10.058. eCollection 2017 Dec. PMID: 29159220

This article present data related to the publication entitled « Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging » (Prevot 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.

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

Vallet-Courbin A, Larivière M, Hocquellet A, Hemadou A, Parimala SN, Laroche-Traineau J, Santarelli X, Clofent-Sanchez G, Jacobin-Valat MJ, Noubhani A.

PLoS One. 2017 Jan 26;12(1):e0170305. doi: 10.1371/journal.pone.0170305. eCollection 2017. PMID: 28125612

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-alphaIIbbeta3 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-alphaIIbbeta3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZalphaA) 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.

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

Hemadou A, Giudicelli V, Smith ML, Lefranc MP, Duroux P, Kossida S, Heiner C, Hepler NL, Kuijpers J, Groppi A, Korlach J, Mondon P, Ottones F, Jacobin-Valat MJ, Laroche-Traineau J, Clofent-Sanchez G. Front Immunol. 2017 Dec 20;8:1796. doi: 10.3389/fimmu.2017.01796. eCollection 2017. PMID: 29326697

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.

5-Solid Lipid Nanoparticles for Image-Guided Therapy of Atherosclerosis.

Oumzil K, Ramin MA, Lorenzato C, Hémadou A, Laroche J, Jacobin-Valat MJ, Mornet S, Roy CE, Kauss T, Gaudin K, Clofent-Sanchez G, Barthélémy P. Bioconjug Chem. 2016 Mar 16;27(3):569-75. doi: 10.1021/acs.bioconjchem.5b00590. Epub 2016 Jan 26.PMID: 26751997

Although the application of nanotechnologies to atherosclerosis remains a young field, novel strategies are needed to address this public health issue. In this context, the magnetic resonance imaging (MRI) approach has been gradually investigated in order to enable image-guided treatments. In this contribution, we report a new approach based on nucleoside-lipids allowing the synthesis of solid lipid nanoparticles (SLN) loaded with iron oxide particles and therapeutic agents. The insertion of nucleoside-lipids allows the formation of stable SLNs loaded with prostacycline (PGI2) able to inhibit platelet aggregation. The new SLNs feature better relaxivity properties in comparison to the clinically used contrast agent Feridex, indicating that SLNs are suitable for image-guided therapy.

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

Jacobin-Valat MJ, Laroche-Traineau J, Larivière M, Mornet S, Sanchez S, Biran M, Lebaron C, Boudon J, Lacomme S, Cérutti M, Clofent-Sanchez G. Nanomedicine. 2015 May;11(4):927-37. doi: 10.1016/j.nano.2014.12.006. Epub 2015 Feb 12. PMID: 25684334

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-alphaIIbbeta3 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.

2014-2012

1-Development of a platform of antibody-presenting liposomes.

Garnier B, Tan S, Gounou C, Brisson AR, Laroche-Traineau J, Jacobin-Valat MJ, Clofent-Sanchez G.

Biointerphases. 2012 Dec;7(1-4):11. doi: 10.1007/s13758-011-0011-9. Epub 2012 Feb 9. PMID: 22589054

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.

2-In vivo phage display to identify new human antibody fragments homing to atherosclerotic endothelial and subendothelial tissues [corrected].

Deramchia K, Jacobin-Valat MJ, Vallet A, Bazin H, Santarelli X, Sanchez S, Dos Santos P, Franconi JM, Claverol S, Bonetto S, Clofent-Sanchez G. Am J Pathol. 2012 Jun;180(6):2576-89. doi: 10.1016/j.ajpath.2012.02.013. Epub 2012 Apr 17. PMID: 22521648

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.

3-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.

Deramchia K, Jacobin-Valat MJ, Laroche-Traineau J, Bonetto S, Sanchez S, Dos Santos P, Massot P, Franconi JM, Martineau P, Clofent-Sanchez G. Int J Mol Sci. 2012;13(6):6902-6923. doi: 10.3390/ijms13066902. Epub 2012 Jun 7. PMID: 22837671

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.

4-The growing interest of fibrin imaging in atherosclerosis.

Clofent-Sanchez G, Jacobin-Valat MJ, Laroche-Traineau J. Atherosclerosis. 2012 May;222(1):22-5. doi: 10.1016/j.atherosclerosis.2012.01.041. Epub 2012 Feb 14. PMID: 22391425

2011-2009

MRI of inducible P-selectin expression in human activated platelets involved in the early stages of atherosclerosis.

Jacobin-Valat MJ, Deramchia K, Mornet S, Hagemeyer CE, Bonetto S, Robert R, Biran M, Massot P, Miraux S, Sanchez S, Bouzier-Sore AK, Franconi JM, Duguet E, Clofent-Sanchez G. NMR Biomed. 2011 May;24(4):413-24. doi: 10.1002/nbm.1606. Epub 2010 Dec 29. PMID: 21192086

The noninvasive imaging of atherosclerotic plaques at an early stage of atherogenesis remains a major challenge for the evaluation of the pathologic state of patients at high risk of acute coronary syndromes. Recent studies have emphasized the importance of platelet-endothelial cell interactions in atherosclerosis-prone arteries at early stages, and the prominent role of P-selectin in the initial loose contact between platelets and diseased vessel walls. A specific MR contrast agent was developed here for the targeting, with high affinity, of P-selectin expressed in large amounts on activated platelets and endothelial cells. For this purpose, PEGylated dextran/iron oxide nanoparticles [PEG, poly(ethylene glycol)], named versatile ultrasmall superparamagnetic iron oxide (VUSPIO) particles, labeled with rhodamine were coupled to an anti-human P-selectin antibody (VH10). Flow cytometry and microscopy experiments on human activated platelets were highly correlated with MRI (performed at 4.7 and 0.2 T), with a 50% signal decrease in T(2) and T(1) values corresponding to the strong labeling of activated vs resting platelets. The number of 1000 VH10-VUSPIO nanoparticles attained per activated platelet appeared to be optimal for the detection of hypo- and hyper-signals in the platelet pellet on T(2) – and T(1) -weighted MRI. Furthermore, in vivo imaging of atherosclerotic plaques in ApoE mice at 4.7 T showed a spatial resolution adapted to the imaging of intimal thickening and a hypo-signal at 4.7 T, as a result of the accumulation of VH10-VUSPIO nanoparticles in the plaque. Our work provides support for the further assessment of the use of VH10-VUSPIO nanoparticles as a promising imaging modality able to identify the early stages of atherosclerosis with regard to the pertinence of both the target and the antibody-conjugated contrast agent used.

2008-2006

1-Identification of human scFvs targeting atherosclerotic lesions: selection by single round in vivo phage display.

Robert R, Jacobin-Valat MJ, Daret D, Miraux S, Nurden AT, Franconi JM, Clofent-Sanchez G. J Biol Chem. 2006 Dec 29;281(52):40135-43. doi: 10.1074/jbc.M609344200. Epub 2006 Oct 26. PMID: 17068330

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE(-/-) mice after in vivo injection. A protein of approximately 56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.

2-Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin.

Robert R, Clofent-Sanchez G, Hocquellet A, Jacobin-Valat MJ, Daret D, Noubhani AM, Santarelli X. Int J Biol Macromol. 2006 Aug 15;39(1-3):51-9. doi: 10.1016/j.ijbiomac.2006.01.014. Epub 2006 Apr 18. PMID: 16620955

Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

 2005-2003

1-Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies.

Robert R, Noubhani AM, Jacobin MJ, Santarelli X, Clofent-Sanchez G. J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Apr 15;818(1):43-51. doi: 10.1016/j.jchromb.2004.10.038. PMID: 15722043

Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.

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

Jacobin MJ, Santarelli X, Laroche-Traineau J, Clofent-Sanchez G. Hum Antibodies. 2004;13(3):69-79. PMID: 15598987

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.

3-Improving selection of alphaIIbbeta3-binding phage antibodies with increased reactivity derived from immunized donors.

Jacobin MJ, Robert R, Pouns O, Laroche-Traineau J, Nurden A, Peter K, Little M, Clofent-Sanchez G. Clin Immunol. 2003 Sep;108(3):199-210. doi: 10.1016/s1521-6616(03)00143-8. PMID: 14499243

Although many studies of the immune response in polytransfused Glanzmann thrombasthenia (GT) patients and in autoimmune thrombocytopenic purpura (AITP) have demonstrated the frequent development of Abs directed against the alphaIIbbeta3 integrin, little is known about the induced anti-alphaIIbbeta3 autoantibodies at the molecular level. Phage display is a powerful technology for selecting and engineering mAbs expressed on the surface of filamentous bacteriophage. Combinatorial libraries of single-chain IgG were constructed from splenocytes from two patients with AITP and one patient with GT. In a previous study, activated platelets or alphaIIbbeta3-expressing CHO cells selection was performed to isolate human IgG anti-alphaIIbbeta3 binding fragments using combinatorial libraries created from the B cells of a GT and an AITP patient. However, we have experienced practical problems such as enrichment of truncated antibodies during selection. We decided to test prolonged treatments with elution agents after screening on the purified form of the alphaIIbbeta3 integrin activated with the RGD peptide. We obtained a higher percentage of clones with full-size antibody fragments as well as an enrichment of more specific alphaIIbbeta3-binding phage-Abs. Some of them, recognizing the activated form of the integrin, would be interesting to further study as potential diagnostic or therapeutic agents in acute coronary syndromes. Sequencing of selected phage-Abs revealed that they used different VH and VL genes with, for the majority of them, a high level of extensive hypermutations in the complementarity determining regions, indicating the diversity of the antigen-driven immune response that occurred in GT and AITP patients.

2002-2000

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

Jacobin MJ, Laroche-Traineau J, Little M, Keller A, Peter K, Welschof M, Nurden A, Clofent-Sanchez G. J Immunol. 2002 Feb 15;168(4):2035-45. doi: 10.4049/jimmunol.168.4.2035. PMID: 11823541

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.

2-Characterisation, cloning and sequencing of a conformation-dependent monoclonal antibody to the alphaIIbbeta3 integrin: interest for use in thrombus detection.

Dabadie M, Valli N, Jacobin MJ, Laroche-Traineau J, Barat JL, Ducassou D, Nurden AT, Clofent-Sanchez G. Platelets. 2001 Nov;12(7):395-405. doi: 10.1080/09537100120071031. PMID: 11674856

The detection of newly formed thrombi is of primary importance in clinical medicine. The activated platelet is a potential target for the localization of thrombotic lesions in arteries. The integrin alpha(IIb)beta(3) membrane changes conformation upon activation. A novel anti-alpha(IIb)beta(3) monoclonal antibody (MAb), XIIF9, is described which recognizes an epitope whose expression was enhanced by activation. Radioiodinated XIIF9 bound to a single class of sites on the beta(3) subunit, with 13600 +/- 2000 molecules bound per unstimulated platelet and a K(d) of 34.5 nM. Platelets stimulated with 0.5 U/ml of thrombin bound 66000 +/- 4000 molecules/cell (K(d) = 51.6 nM). Moreover, XIIF9 binding to unstimulated platelets could be increased 4-fold by treatment of the alpha(IIb)beta(3) complex with 5 mM EDTA. Thus, XIIF9 recognized an epitope on the beta(3) subunit whose accessibility was increased upon thrombin activation or EDTA treatment. Sequence analysis of the gene segment encoding the XIIF9 heavy chain revealed interesting motifs shared with cyclic CX6-7C anti-alpha(IIb)beta(3) peptides or with AC7, a published MAb specific for activated alpha(IIb)beta(3). In vivo experiments in atherosclerotic rabbits followed by immunohistological analysis, revealed a specific binding of XIIF9 on platelets engaged in thrombus formation, demonstrating real clinical potential for such MAbs in imaging.

1999-1992

1-Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments.

Laroche-Traineau J, Jacobin MJ, Biard-Piechaczyk M, Vuillemin L, Chagnaud JL, Pau B, Nurden AT, Clofent-Sanchez G. FEBS Lett. 1999 Oct 22;460(1):86-92. doi: 10.1016/s0014-5793(99)01308-3. PMID: 10571066

A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen-binding specificity of the parental mAb.

2-Analysis of VH and VL genes of a monospecific human anti-myosin antibody produced by a B cell from the primary repertoire.

Laroche-Traineau J, Biard-Piechaczyk M, Jacobin MJ, Chagnaud JL, Pau B, Nurden A, Clofent-Sanchez G. Hum Antibodies. 1999;9(3):177-88. PMID: 10690632

Epstein-Barr virus (EBV) transformation of B lymphocytes from a Glanzmann’s thrombasthenia patient with a serum antibody to the integrin alpha IIb beta 3, led to the immortalization of a B cell secreting a monospecific IgM monochonal antibody (MAb), B7, reactive with platelet myosin. Analysis of B7 V genes revealed minimally mutated sequences: the immortalized B cell issued from the primary repertoire, with no evidence of an in vivo selection by myosin. The V genes were here compared with sequences of human MAbs available on databases to more clearly understand the monospecificity of the B7 MAb. B7 V genes were closely identical to rearranged V genes in clones with self-specificities, often secreting polyreactive antibodies. In contrast, B7 is an unmutated monoreactive human MAb able to recognize myosin with a high avidity. Comparison of the CDR3H sequence with that of MAbs in databases supports a central role for the CDR3H subdomain in determining monospecificity. Our results suggest the existence of a monospecific autoreactive B cell compartment, besides the well-known polyspecific one, susceptible to be the template of pathogenic autoreactivity, characterized by antibodies of high affinity and specificity.