2024-2021

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.

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.

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

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.

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.

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.

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.

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

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.

Data on iron oxide core oil-in-water nanoemulsions for atherosclerosis imaging.

Prévot G, Mornet S, Lorenzato C, Kauss T, Adumeau L, Gaubert A, Baillet J, Barthélémy P, Clofent-Sanchez G, Crauste-Manciet S.

Data Brief. 2017 Oct 26;15:876-881. doi: 10.1016/j.dib.2017.10.059. eCollection 2017 Dec.

PMID: 29159224

The data presented in this article are 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 synthesis and the characteristics of the Superparamagnetic Iron Oxide Nanoparticles (SPION) loaded inside nanoemulsions (NEs). Focus was set on obtaining SPION with narrow size distribution and close to superparamagnetic limit (20 nm) in order to reach a reasonable magnetic signal. Nanoparticles (NPs) of three different sizes were obtained (7, 11 and 18 nm) and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), diffuse reflectance infrared Fourier transform (DRIFT) and thermogravimetric analysis (TGA). SPION were coated with oleic acid (OA) in order to load them inside the oily core of NEs droplets. SPION loaded NEs were magnetically sorted using MACS(R) MS Column (Miltenyi Biotec) and iron quantification was performed by UV-spectrometry measurements.

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.

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.

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode.

Pourtau L, Sellal JM, Lacroix R, Poncelet P, Bernus O, Clofent-Sanchez G, Hocini M, Haïssaguerre M, Dignat-George F, Sacher F, Nurden P.

Int J Cardiol. 2017 Sep 15;243:216-222. doi: 10.1016/j.ijcard.2017.03.068.PMID: 28747025

BACKGROUND: Thrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode. METHODS: Blood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, alphaIIbbeta3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays. RESULTS: A specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed. CONCLUSION: Acute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization.

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.

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.

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

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.

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.

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.

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.

Nanoparticle phagocytosis and cellular stress: involvement in cellular imaging and in gene therapy against glioma.

Bouzier-Sore AK, Ribot E, Bouchaud V, Miraux S, Duguet E, Mornet S, Clofent-Sanchez G, Franconi JM, Voisin P.

NMR Biomed. 2010 Jan;23(1):88-96. doi: 10.1002/nbm.1434. PMID: 19795366

In gene therapy against glioma, targeting tumoral tissue is not an easy task. We used the tumor infiltrating property of microglia in this study. These cells are well adapted to this therapy since they can phagocyte nanoparticles and allow their visualization by MRI. Indeed, while many studies have used transfected microglia containing a suicide gene and other internalized nanoparticles to visualize microglia, none have combined both approaches during gene therapy. Microglia cells were transfected with the TK-GFP gene under the control of the HSP(70) promoter. First, the possible cellular stress induced by nanoparticle internalization was checked to avoid a non-specific activation of the suicide gene. Then, MR images were obtained on tubes containing microglia loaded with superparamagnetic nanoparticles (VUSPIO) to characterize their MR properties, as well as their potential to track cells in vivo. VUSPIO were efficiently internalized by microglia, were found non-toxic and their internalization did not induce any cellular stress. VUSPIO relaxivity r(2) was 224 mM(-1).s(-1). Such results could generate a very high contrast between loaded and unloaded cells on T(2)-weighted images. The intracellular presence of VUSPIO does not prevent suicide gene activity, since TK is expressed in vitro and functional in vivo. It allows MRI detection of gene modified macrophages during cell therapy strategies.

Thrombocytopenia after abciximab use results from different mechanisms.

Lajus S, Clofent-Sanchez G, Jais C, Coste P, Nurden P, Nurden A.

Thromb Haemost. 2010 Mar;103(3):651-61. doi: 10.1160/TH09-08-0603. Epub 2010 Jan 13. PMID: 20076853

Our study concerns thrombocytopenia in patients with acute ischaemic coronary artery disease receiving anti-platelet drugs to the aIIbb3 integrin (GPIIb/IIIa). We have screened for drug-dependent antibodies (DDAB) in 18 patients who suffered a fall of > 50% in platelet count (9 patients had a nadir of <50,000 platelets/microl) after receiving abciximab and related results to clinical outcome. Serum or plasma was screened for DDAB using (i) a direct ELISA against purified aIIbb3, aIIbb3-abciximab complexes or abciximab alone, (ii) control platelets and flow cytometry and (iii) monoclonal antibody immobilisation of platelet antigens. DDAB were found for 11 patients, with aIIbb3 ELISA the most sensitive test. Progressive platelet consumption linked with haemoglobin loss and/or use of intra-aortic balloon pumping, another potential cause of a fall in platelet count, was also evaluated. DDAB were identified that recognised aIIbb3 associated with abciximab and/or abciximab alone. Screening of both progressive and delayed thrombocytopenia (appearing after 5 to 11 days) suggested that antibodies against abciximab preceded those recognising neo-epitopes on aIIbb3, with a time-dependent broadening of antibody specificities. Higher titres were seen after second abciximab use. Five antibodies were platelet-activating. In conclusion, the mechanisms responsible for this complication of anti-aIIbb3 therapy are multiple and often associated with a complex immune response.

Rapid screening of purification strategies for the capture of a human recombinant F(ab’)2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS.

Pezzini J, Brenac Brochier V, Barrouillet MP, Cerruti M, Clofent-Sanchez G, Schapman A, Topol A, Robert R, Cabanne C, Cerruti P, Santarelli X.

J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Aug 15;877(24):2428-34. doi: 10.1016/j.jchromb.2009.04.034. Epub 2009 May 4.PMID: 19467934

The development of a capture step of a human recombinant F(ab’)(2) produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab’)(2) was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.

2008-2006

A case of profound and prolonged tirofiban-induced thrombocytopenia and its correction by intravenous immunoglobulin G.

Clofent-Sanchez G, Harizi H, Nurden A, Coste P, Jais C, Nurden P.

J Thromb Haemost. 2007 May;5(5):1068-70. doi: 10.1111/j.1538-7836.2007.02440.x.

PMID: 17461936

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.

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

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.

Delayed immunologic thrombocytopenia induced by abciximab.

Nurden P, Clofent-Sanchez G, Jais C, Bermejo E, Leroux L, Coste P, Nurden AT.

Thromb Haemost. 2004 Oct;92(4):820-8. doi: 10.1160/TH04-04-0237. PMID: 15467914

Abciximab is an anti-GPIIb-IIIa drug widely used to prevent thrombotic complications during percutaneous coronary intervention. We now report on the immunologic origin of thrombocytopenia developing between 7 and 12 days after the onset of abciximab infusion. Antibodies directed against abciximabcoated platelets were located in 5 patients with delayed thrombocytopenia, just as they were present in a patient whose platelet count fell within a few hours after receiving the drug. Abciximab-dependent IgG antibody was revealed in serum using control platelets in the monoclonal antibody immobilization of platelet antigens assay (MAIPA) performed with SZ22, a MoAb to GPIIb. The presence of IgG antibodies specific for platelets sensitized with abciximab was confirmed by flow cytometry. They were not located in 13 patients receiving abciximab but whose platelet counts remained stable. For three patients, antibodies were transient and their presence related to the extent of the thrombocytopenia. Surprisingly, antibodycontaining plasma from three patients induced abciximabdependent activation and aggregation of normal platelets, a finding confirmed by electron microscopy. Immunogold labeling revealed that abciximab was associated with platelets in the aggregate, suggesting that its inhibitory effect was overcome by the platelet stimulation. In summary, these results show that abciximab-dependent thrombocytopenia can be delayed and potentially prothrombotic.

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.

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

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.

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.

Three-step purification of bacterially expressed human single-chain Fv antibodies for clinical applications.

Laroche-Traineau J, Clofent-Sanchez G, Santarelli X.

J Chromatogr B Biomed Sci Appl. 2000 Jan 14;737(1-2):107-17. doi: 10.1016/s0378-4347(99)00441-7. PMID: 10681047

We have obtained a cell line which secretes a human monoclonal IgM (B7) reacting with the myosin heavy chain of human heart. We have constructed single-chain fragments (scFv) of B7. The scFv may be useful for the imaging of myocardial necrosis after myocarditis, cardiac drug toxicosis or graft rejection. The aim of our work was to purify the scFv for immunoscintigraphy. We describe several purification steps including immobilized metal affinity chromatography (IMAC), anti-c-myc monoclonal antibody affinity chromatography, size-exclusion chromatography with Superdex 75 HR 10/30 and ion-exchange chromatography (mini Q TM 30Q).

1999-1992

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.

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.

Three cases of acquired von Willebrand disease associated with systemic lupus erythematosus.

Viallard JF, Pellegrin JL, Vergnes C, Borel-Derlon A, Clofent-Sanchez G, Nurden AT, Leng B, Nurden P.

Br J Haematol. 1999 May;105(2):532-7. PMID: 10233433

Acquired von Willebrand disease associated with systemic lupus erythematosus (SLE) has been detected in three middle-aged women. In each case the first clinical manifestation was a bleeding syndrome. Plasma levels of von Willebrand factor (VWF) and ristocetin-induced platelet agglutination were as found in type 1 von Willebrand disease for the first patient, type 3 for the second patient, and type 2 for the third patient. Intraplatelet levels of VWF were normal for all three patients. In all cases a mixture of patient’s plasma with normal plasma resulted in inhibition of ristocetin-induced binding of VWF to normal platelets. Intravenous immunoglobulin given to patients 2 and 3 corrected the plasma VWF level of the second patient but not that of the third. Therapy with corticosteroids was partially beneficial for patient 3 and patient 2. For patient 2, the severity of the cutaneous lesions also led to the use of cyclophosphamide, and this therapy resulted in total correction of VWF levels. Our observations confirm previous reports of acquired von Willebrand syndrome associated with SLE and show heterogeneity both in the phenotypic form and in the response to treatment.

Characterization and application of new macroporous membrane ion exchangers.

Santarelli X, Domergue F, Clofent-Sanchez G, Dabadie M, Grissely R, Cassagne C.

J Chromatogr B Biomed Sci Appl. 1998 Feb 27;706(1):13-22. doi: 10.1016/s0378-4347(97)00532-x. PMID: 9544803

A new ready-to-use unit for high-performance membrane chromatography has been characterized. Its dynamic capacity, resolving power and protein recovery were measured at different flow-rates. The binding capacity was 0.5-2 mg/cm2 with a 95% recovery at 10 ml/min irrespective of the protein concentration up to 10 mg/ml. For very-high flow-rates (50 and 100 ml/min) the recovery was 90% and 70%. At these flow-rates, the maximum back-pressure was about 0.1 MPa and was independent of the filtration area. By increasing the filtration area, a proportional capacity increase was obtained, indicating an easy scale-up. High flow-rates had only a slight effect on resolution. This new adsorber was able to purify IgM from supernatant of cell culture of a human hybridoma in less than 8 min with a high degree of purity (95%).

Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb-IIIa in a patient with an unusual platelet membrane glycoprotein composition.

Macchi L, Nurden P, Marit G, Bihour C, Clofent-Sanchez G, Combrié R, Nurden AT.

Am J Hematol. 1998 Feb;57(2):164-75. doi: 10.1002/(sici)1096-8652(199802)57:2<164::aid-ajh13>3.0.co;2-c. PMID: 9462551

The subject (E.B.) is a 63-year-old woman with autoimmune thrombocytopenic purpura (AITP) who was first examined some 6 years ago with symptoms of epistaxis and gum bleeding, severe thrombocytopenia, and large platelets. Her serum tested positively with control platelets in the MAIPA assay performed using monoclonal antibodies (MoAb) to glycoprotein (GP) IIIa (XIIF9, Y2/51), yet was negative in the presence of MoAbs to GP IIb (SZ 22) or to the GP IIb-IIIa complex (AP2, P2). The patient’s platelets failed to aggregate with all agonists tested except for ristocetin. IgG isolated from the patient’s serum inhibited ADP-induced aggregation of control platelets. Unexpectedly, flow cytometry showed an altered expression of membrane glycoproteins on the patient’s platelets. Levels of GP Ib-IX were much higher than previously located by us in platelets. In contrast, the expression of GP IIb-IIIa was about half that seen with control subjects. When Western blotting was performed, a striking finding was a strong band of 250 kDa recognized by a series of MoAbs to GP Ib alpha in addition to the band in the normal position of GP Ib alpha. Finally, ADP-stimulated (E.B.) platelets failed to express activation-dependent epitopes on GP IIb-IIIa as recognized by PAC-1, AP6, or F26 and additionally gave a reduced P-selectin expression after thrombin addition. In conclusion, we present a novel patient with a severely perturbed platelet function where an altered membrane GP profile is associated with the presence of an autoantibody recognizing a complex-dependent determinant on GP IIb-IIIa and inhibitory of platelet aggregation.

Anti-platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia.

Macchi L, Rispal P, Clofent-Sanchez G, Pellegrin JL, Nurden P, Leng B, Nurden AT.

Br J Haematol. 1997 Aug;98(2):336-41. doi: 10.1046/j.1365-2141.1997.2243038.x. PMID: 9266930

The role of antiphospholipid antibodies in the pathogenesis of the thrombocytopenia observed during primary antiphospholipid antibody syndrome (APAS) and systemic lupus erythematosus (SLE) remains controversial. We have used the MAIPA test to examine the frequency and specificity of anti-platelet antibodies directed against the major platelet membrane glycoproteins (GP IIb-IIIa, GP Ib-IX, GP Ia-IIa and GP IV) in patients where SLE and APAS were associated or not with thrombocytopenia. Results were compared with a series of 26 ITP patients, 46% of whom were shown to possess anti-platelet antibodies directed against one or more of the platelet surface glycoproteins. When APAS was associated with thrombocytopenia, 7/10 patients possessed antibodies against GP IIb-IIIa and/or GP Ib-IX. For SLE patients with thrombocytopenia, 6/10 patients were shown to have antiplatelet antibodies against GP IIb-IIIa, GP Ib-IX or GP IV. In contrast, for APAS (n=11) and SLE patients (n=11) without thrombocytopenia, only one patient had an antibody directed against GP IIb-IIIa and one patient had an antibody to GP IV. Our results suggest that antibodies directed against major platelet membrane glycoproteins may play a role in the thrombocytopenia that is seen during SLE and APAS.

Incidence of anti-mouse antibodies in thrombocytopenic patients with autoimmune disorders.

Clofent-Sanchez G, Laroche-Trainean J, Lucas S, Rispal P, Pellegrin JL, Nurden P, Nurden A.

Hum Antibodies. 1997;8(2):50-9. PMID: 9289388

Idiopathic thrombotycopenic purpura (ITP) is an autoimmune disorder in which circulating autoantibodies react with target antigens on the platelet membrane. In order to identify the autoimmune response in ITP, two MAIPA (Monoclonal Antibody (MAb) Immobilization of Platelet Antigen) assays (MAIPA I and MAIPA II) were performed on sera from thrombocytopenic patients. In the classic MAIPA assay (MAIPA I), control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein. In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A positive MAIPA I test but a negative MAIPA II has been shown to result from the presence of serum antibodies recognizing mouse MAb to platelet glycoproteins used in the assay. We compared the frequency of such ‘anti-mouse’ antibodies in patients with thrombocytopenia associated or not with other autoimmune states and in healthy donors with a normal platelet count. Statistically significant differences were found in the incidence of anti-mouse antibodies between patients and healthy donors. Furthermore, the identity of the targeted mouse MAbs varied in sera from the patients. The detected anti-mouse antibodies may include anti-idiotypic antibodies produced against cross-reactive idiotypes shared by human and mouse anti-platelet antibodies.

PAICA: a method for characterizing platelet-associated antibodies–its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3.

Macchi L, Clofent-Sanchez G, Marit G, Bihour C, Durrieu-Jais C, Besse P, Nurden P, Nurden AT.

Thromb Haemost. 1996 Dec;76(6):1020-9. PMID: 8972027

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA (« Platelet-Associated IgG Characterization Assay »), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient’s own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA (« Monoclonal Antibody-specific Immobilization of Platelet Antigens »). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

A close spatial relationship between GP IIb-IIIa complexes and CD9 antigen as demonstrated by the MAIPA technique.

Laroche-Traineau J, Macchi L, Marit G, Nurden P, Nurden AT, Clofent-Sanchez G.

Platelets. 1996;7(5-6):303-11. doi: 10.3109/09537109609023593. PMID: 21043666

CD9 is a well-defined component of the platelet plasma membrane and has a copy number almost equivalent to that of glycoprotein (GP) IIb-IIIa complexes, the aggregation receptor on platelets. It has an apparent molecular mass of 24 kD and is otherwise known as p24. Stimulation of p24 by monoclonal antibodies (MAb) induces platelet aggregation and granule release, involves FcgammaRII, and is mainly mediated through the stimulation of phospholipase C. In accordance with a signalling function, p24 has been reported to associate with small GTP-binding proteins and to GP IIb-IIIa complexes upon activation. We now report further evidence of a strong relationship between p24 and GP IIb-IIIa in platelets. Using the MAIPA (monoclonal antibody immobilization of platelet antigens) assay in the screening of human antibodies to platelet glycoproteins, we found that GP IIb-IIIa-antibody complexes were almost invariably associated with p24 in the harvested detergent-soluble fraction of platelet lysates. Thus, associated human antibodies were detected following the targeting of either GP IIb-IIIa or p24 by monospecific murine monoclonal antibodies (MAbs). This is a point to bear in mind when assessing for antibodies to p24 or GP IIb-IIIa in immune thrombocytopenias.

Autoantibodies and anti-mouse antibodies in thrombocytopenic patients as assessed by different MAIPA assays.

Clofent-Sanchez G, Lucas S, Laroche-Traineau J, Rispal P, Pellegrin JL, Nurden P, Nurden A.

Br J Haematol. 1996 Oct;95(1):153-60. doi: 10.1046/j.1365-2141.1996.d01-1888.x. PMID: 8857954

Two MAIPA (monoclonal antibody [MAb] immobilization of platelet antigen) assays were performed to determine (a) autoantibodies to platelet glycoproteins (GP) and (b) serum antibodies recognizing mouse MAbs used in the assay. In MAIPA I, control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein (GP IIb-IIIa, Ib-IX, Ia-IIa, IV and p24). In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A series of 25 patients with autoimmune thrombocytopenic purpura (ATP) associated or not with other autoimmune states were examined. Autoantibodies (both MAIPA I and MAIPA II positive) or anti-mouse Abs (MAIPA I positive and MAIPA II negative) were frequent in both groups of patients. Statistically significant differences existed in the incidence of anti-mouse Abs between patients (56.5%) and healthy donors (10%). This suggests that their production may be related to thrombocytopenias associated with autoimmune disease. We speculate that the presence of anti-mouse antibodies could reflect an abnormality in the immunological modulation of the idiotypic network.

A human monoclonal antibody obtained from EBV-transformed B cells with specificity for myosin.

Laroche-Traineau J, Clofent-Sanchez G, Daret D, Bonnaud E, Barat JL, Ducassou D, Nurden AT.

Br J Haematol. 1995 Dec;91(4):951-62. doi: 10.1111/j.1365-2141.1995.tb05419.x. PMID: 8547148

We describe the preparation of a stable human lymphoblastoid cell line obtained during ex vivo studies in which peripheral blood lymphocytes of a Glanzmann’s thrombasthenia patient were transformed with Epstein-Barr virus. Somatic hybrids secreted an IgM monoclonal antibody (B7) that reacted with the myosin heavy chain of human platelets by immunoblotting. Flow cytometry showed that B7 barely recognized unstimulated intact platelets, but bound abundantly after permeabilization of fixed cells with Triton X-100. The reactivity of the antibody on thin sections of human myocardium and aorta was studied by immunohistochemistry. B7 specifically stained myosin of myocytes, but there was no labelling of aortic smooth muscle cells. The epitope was conserved in cardiac or skeletal myosin prepared from pig or rabbit. Measurement of the dissociation constant in a competitive ELISA showed that B7 bound with high affinity (10(-8) M). Purified Fab fragments retained their ability to bind to myosin, suggesting that B7 may be useful in the imaging of myocardial necrosis after myocardial infarction, myocarditis, cardiac drug toxicosis or graft rejection. This work also shows that EBV transformation of B cells may uncover naturally occurring autoantibodies which under normal circumstances are inhibited by the immune surveillance system.

A patient with autoimmune thrombocytopenic purpura demonstrating serum antibodies reactive with mouse cross-reactive idiotypes.

Clofent-Sanchez G, Laroche-Traineau J, Bermudes H, Lucas S, Nurden P, Nurden A.

Clin Immunol Immunopathol. 1995 Dec;77(3):271-81. doi: 10.1006/clin.1995.1153. PMID: 7586737

Autoimmune thrombocytopenic purpura (ATP) is a syndrome of destructive thrombocytopenia due to platelet-binding antibodies. We report the case of a young woman (C.V.) who has a history of chronic ATP with severe but transient bouts of thrombocytopenia. Using the classic monoclonal antibody (MAb) immobilization of platelet antigens (MAIPA) assay to screen serum antibody specificity, results were strongly positive with MAbs to glycoproteins (GPs) Ib-IX, Ia-IIa, IV, and p24, but weakly positive or negative for GP IIb-IIIa. In contrast, a two-step incubation assay (MAIPA II), in which platelets were incubated sequentially with C.V. serum and the murine MAb, gave negative results for all GPs. Affinity chromatography performed using Bx-1, a MAb to GP Ib, showed that the patient’s serum contained antibodies to determinants expressed by mouse immunoglobulins. These were present on Fab fragments on Bx-1. A survey of sera from other patients with thrombocytopenia of immune origin revealed that antibodies reactive with selected idiotypes of mouse MAbs were not infrequent and raises the question of their role in the thrombocytopenia.

The ex vivo production of human monoclonal antibodies to glycoprotein IIb-IIIa complexes of blood platelets.

Laroche-Traineau J, Clofent-Sanchez G, Vezon G, Nurden AT.

Hum Antibodies Hybridomas. 1994;5(3-4):165-77. PMID: 7538808

The integrin alpha IIb beta 3 (GPIIb-IIIa complex) of blood platelets mediates platelet aggregation by binding adhesive proteins which form bridges between activated cells. This same process is implicated in arterial thrombosis. The goal of our research is to take B-lymphocytes from patients possessing inhibitory antibodies to GPIIb-IIIa and develop technology permitting their production ex vivo. Starting point is the peripheral blood from two patients with Glanzmann’s thrombasthenia, an inherited disorder in which platelets lack these complexes, and where high titre antibodies to GPIIb-IIIa have formed following contact with normal platelets after transfusion and/or pregnancy. We describe a strategy of in vitro stimulation to overcome the following constraints: (i) peripheral blood contains a low concentration of antigen-reactive specific B-cells, and (ii) the circulating B-cells are arrested in a phase in which additional stimuli are required to induce antigen-specific clonal activation. Optimal conditions involve the use of a combination of growth factors, polyclonal activators and soluble GPIIb-IIIa prior to the fusion of activated B-cells with either (a) the murine myeloma cell line X63 Ag 8,653 or (b) the heteromyeloma cell line SPM4-0. In this way, we have obtained several cell lines secreting antibodies specific for the GPIIb-IIIa complex. Our next aim is to rescue the relevant human immunoglobulin genes from these hybridoma cells.

Further evidence that heparin-dependent thrombocytopenia may result from Fc receptor-mediated interactions.

Laroche J, Clofent-Sanchez G, Jallu V, Hourdillé P, Vezon G, Nurden AT.

Nouv Rev Fr Hematol (1978). 1992;34(1):111-21. PMID: 1523092

Heparin-dependent thrombocytopenia (HDT) and associated thrombotic complications, are thought to be linked to the appearance of anti-platelet antibodies. Attempts were made to characterize the antibodies in the sera from 10 such patients. Western blotting against platelet antigens was inconclusive, often revealing multiple bands but with considerable variability from patient to patient. An often seen band of approximately 83 kDa was also given by some nonimmune sera and the antigen appeared to be predominantly intracellular in origin. Antibodies to major membrane glycoproteins were not readily apparent. This was confirmed by the MAIPA (« Monoclonal Antibody Immobilization of Platelet Antigens ») test, performed to detect antibodies to GP Ib-IX and GP IIb-IIIa. Only one weak activity to GP Ib-IX and one weak activity to GP IIb-IIIa were detected. In contrast, an antibody to the PlA1 alloantigen was readily detected in the serum of an additional patient. The ability of HDT serum to induce the aggregation of control platelets was studied in detail. Aggregation was inhibited by EDU-3, a monoclonal antibody to GP IIb-IIIa complexes, and by the synthetic peptide RGDS, suggesting an involvement of the same pathway as used by physiologic agonists. However, in agreement with Chong et al (Thromb Res 55:291, 1989), we observed that aggregation was inhibited by rabbit IgG, suggesting that it was Fc-receptor mediated. Interestingly, 2E1, a monoclonal antibody to the Fc gamma RII receptor, induced platelet aggregation with a lag phase, a characteristic of HD antibodies. Nonetheless, for different donors, there was no correlation between the length of the lag phase induced by 2E1 and HD antibodies. Fc-receptor blockade should be considered as a means for diminishing the clinical complications of HDT.

Analysis of the B-cell compartment in plasma cell leukemia and multiple myeloma: immunoglobulin gene rearrangement of EBV-infected B-cell lines.

Commes T, Clofent G, Ghanem N, Zhang XG, Lefranc MP, Bataille R, Klein B.

Leukemia. 1993 Apr;7(4):609-17.

PMID: 7681918Multiple myeloma (MM) is defined as a tumoral expansion of plasma cells occurring in the bone marrow and sometimes in the peripheral blood (plasma-cell leukemia, PCL). Many reports have demonstrated a clonal expansion of B cells bearing the same idiotypic determinants as the myeloma protein (idiotypic B cells) in MM, suggesting that they could belong to the malignant clone. In order to investigate whether the B-cell population is a malignant component or not, either in the peripheral blood of patients with PCL or in the bone marrow of patients with MM, we derived B-cell lines by infecting, with the Epstein-Barr virus (EBV), cultures in limiting dilution of mononuclear cells from six patients. A limiting dilution culture was used to prevent the elimination of slowly proliferating clones by the more rapidly dividing ones, and thus to get the most exact representation of the B-cell repertoire of these patients. The cloning efficiency of the EBV-infected cells was similar in patients and healthy individuals (range: 1 in 100 to 1 in 1650 B cells). All of the clones obtained from a single patient exhibited different clonal immunoglobulin gene rearrangements (IGR), proving the validity of our cloning technique. No tumoral clones (61 clones analysed) showed the IGR pattern specific of autologous myeloma cells. These results indicate that malignant plasma cells cannot be immortalized with EBV. These results show that, if malignant B cells (pre-switch or post-switch) exist, they could be present only in a minor population, and the corollary of this is that there is a major population of non-malignant B cells in the sites of tumoral proliferation of patients with MM. This is remarkable in view of numerous reports showing a profound defect of the polyclonal B lymphopoiesis in these patients, and even an absence of B lymphocytes. Thus, these results challenge the existence of a major compartment of malignant idiotypic B cells and favor the hypothesis of non-malignant B cells sharing cross-reactive idiotypes with the autologous myeloma protein.

1991-1989

Human natural killer cells suppress the proliferation of B cells.

Commes T, Clofent G, Jourdan M, Bataille R, Klein B.

Immunol Lett. 1990 Mar-Apr;24(1):57-61. doi: 10.1016/0165-2478(90)90036-p. PMID: 2142675

We have investigated the suppressive effect of human natural killer (NK) cells on autologous B-cell proliferation. Removal of NK cells by anti-NK-cell monoclonal antibodies (CD16, Leu 11b; Leu 7) increased by 2-3-fold the proliferative response of purified B cells activated by anti-mu and B-cell growth factor (BCGF). The inhibitory effect of NK cells was observed using recombinant IL-2 or semi-purified BCGF-I as sources of BCGF. Moreover NK cells, highly purified by centrifugation on a Percoll discontinuous density gradient, suppressed the proliferative response of purified autologous B cells activated by anti-mu and BCGF. These results show a suppressive effect of human NK cells on B-cell proliferation in vitro.

The defect in peripheral blood B-cell activation in patients with multiple myeloma is not due to a deficiency in the production of B-cell growth and differentiation factors.

Commes T, Klein B, Jourdan M, Clofent G, Houssiau F, Grenier J, Bataille R.

J Clin Immunol. 1989 Jan;9(1):65-73. doi: 10.1007/BF00917129. PMID: 2495299

The suppression of B lymphopoiesis is a major feature of multiple myeloma (MM). In this disease, there is a striking defect in the response of peripheral blood B cells to pokeweed mitogen (PWM). Normally, B-cell activation depends on B-cell growth factors (BCGFs) and B-cell differentiation factors (BCDFs), produced by peripheral blood mononuclear cells. We therefore evaluated whether the production of these cytokines was defective in patients with MM. We have studied the production of BCGFs (using the anti-mu assay) and, particularly, interleukin-2 and interferon-gamma, two well-documented BCGFs. No defect in the production of BCGFs, interleukin-2, and interferon-gamma was found in patients with active (N = 14) or stable (N = 10) MM, compared with healthy donors (N = 13). The production of BCDFs (i.e., overall activity) was also evaluated and, more particularly, that of interleukin-6 (IL-6). This cytokine is a potent BCDF which is essential in the PWM-induced activation of B cells, acting at the terminal stages of B-cell differentiation. Again, no defect in the production of BCDFs and IL-6 was found in patients with MM. Therefore, the ability to secrete cytokines controlling the process of B-cell activation is not affected in such patients. This indicates that the profound failure of humoral immune response is not due to deficiency of peripheral blood mononuclear cells producing these factors.

Limiting dilution cloning of B cells from patients with multiple myeloma: emergence of non-malignant B-cell lines.

Clofent G, Klein B, Commes T, Vincent C, Ghanem N, Lenoir G, Lefranc MP, Bataille R.

Int J Cancer. 1989 Apr 15;43(4):578-86. doi: 10.1002/ijc.2910430408. PMID: 2539329

Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of slowly proliferating malignant plasma cells in the bone marrow (BM). Several reports have shown the existence of an abnormal B-cell compartment including proliferative idiotypic B cells (i.e., B cells bearing the same idiotypic determinants as the myeloma protein) in the BM and peripheral blood (PB) of patients with MM. In order to study whether this abnormal compartment can be grown in vitro, we cultured the PB and BM of 23 patients with MM using limiting dilution methods. Our purpose was to restrict the effect of suppressor cells and the possible overgrowth of the cultures by the more rapidly growing B cells, which occurs in bulk cultures. Spontaneously growing cells were obtained only from patients seropositive for the Epstein-Barr virus (EBV) and all the cultures were composed of B cells carrying the EBV genome. Thus, positive cultures were generated only in the presence of B cells latently infected with EBV in vivo. The mean frequency of these B cells (1 in 25,000 B cells) was as low in MM patients as in healthy donors. This low frequency indicated that malignant cells do not bear the EBV genome in vivo and that the in vivo regulation of the EBV infection is unaffected in patients with MM. No Ig-gene rearrangements, specific of the autologous myeloma cells, were found in the cell lines obtained from BM or PB. Thus, the putative malignant B cells or myeloma cells were not able to generate cell lines in vitro, either spontaneously or after endogenous infection with EBV.

No detectable malignant B cells in the peripheral blood of patients with multiple myeloma.

Clofent G, Klein B, Commes T, Ghanem N, Lefranc MP, Bataille R.

Br J Haematol. 1989 Mar;71(3):357-61. doi: 10.1111/j.1365-2141.1989.tb04292.x. PMID: 2649139

Previous studies have reported the presence of idiotypic B lymphocytes in the peripheral blood of patients with multiple myeloma (MM), suggesting that they may belong to the malignant clone. This led us to investigate by Southern blot analyses the presence of tumour-specific immunoglobulin-gene (Ig-gene) rearrangements in the peripheral-blood mononuclear cells of 21 MM patients. This method was shown to detect clonal cells when they represent as little as 2% of the cell population. B-cell-enriched fractions were also studied in nine cases. An occasional contamination by circulating malignant plasma cells was carefully evaluated using immunofluorescence. Clonal rearrangements were observed in only two cases, in which a contamination by myeloma cells was evident. In these cases the use of different endonucleases clearly demonstrated that these Ig-clonal rearrangements involved post-switched cells. No clonal rearrangement was found when contamination by myeloma cells was absent. Our results demonstrate the absence of detectable B cells involved in the myeloma clone in the peripheral blood of patients with MM.

No preferential use of the VH(V) family in human multiple myeloma.

Clofent G, Brockly F, Commes T, Lefranc MP, Bataille R, Klein B.

Br J Haematol. 1989 Dec;73(4):486-90. doi: 10.1111/j.1365-2141.1989.tb00285.x. PMID: 2611136

A recently described immunoglobulin VH family (the VH(V) family) close to the DH and JH genes is preferentially rearranged in immature B-cell tumours. The question of the emergence of multiple myeloma (MM) from a tumorous pre-B cell is not yet resolved. To draw a comparison with chronic lymphocytic leukaemia (CLL), we studied the VH(V) rearrangements in 28 MM patients. A rearranged Hind III-Bam HI fragment of 9.5 kb was detected in only one patient instead of the rearranged fragment of 8.5 kb described in CLL. Rearrangements of a member of the VH(V) family in a 9.5 kb fragment were also observed in two out of 20 lymphoblastoid cell lines obtained from peripheral blood of MM patients. We report here that the VH(V) family is not preferentially involved in this pathology and that the size of the only rearrangement obtained is larger than the 8.5 kb fragment observed in CLL. These results do not favour the hypothesis of a pre-B cell involvement in MM.

Purine metabolizing enzymes of lymphocyte cell populations: correlation between AMP-deaminase activity and dATP accumulation in murine lymphocytes.

Dornand J, Clofent G, Bonnafous JC, Favero J, Mani JC.

Proc Soc Exp Biol Med. 1985 Sep;179(4):448-55. doi: 10.3181/00379727-179-42122. PMID: 2991938