16 research outputs found
Discovery of human antibodies and identification of new biomarkers for atheroma targeting
Cette thĂšse porte sur la sĂ©lection dâanticorps humains et la dĂ©couverte de nouveaux biomarqueurs en vue dâun diagnostic et dâune thĂ©rapie de lâathĂ©rosclĂ©rose. Le premier volet concerne, dans un premier temps, la sĂ©lection dâanticorps humains dans un contexte physiopathologique chez un modĂšle animal de lâathĂ©rosclĂ©rose. La sĂ©lection in vivo a permis de sĂ©lectionner des anticorps sur large pannel de cibles dans leur conformation native et sous lâinfluence de leur microenvironnement. Suite Ă cela, afin dâaccĂ©der Ă la spĂ©cificitĂ© des clones, une mĂ©thode innovante de criblage a Ă©tĂ© dĂ©veloppĂ©e, la cytomĂ©trie de flux Ă moyen dĂ©bit. Cette technique, contrairement Ă lâELISA couramment utilisĂ© pour la caractĂ©risation dâanticorps ou de peptides, ne nĂ©cessite pas de connaitre lâantigĂšne et permet Ă©galement de travailler Ă des concentrations de protĂ©ines moindres. Ainsi, deux cents anticorps ont pu ĂȘtre sĂ©lectionnĂ©s. Les tests dâaffinitĂ© et de caractĂ©risation de ces anticorps ont, par la suite, menĂ© Ă lâidentification dâune nouvelle cible de la plaque dâathĂ©rome, la galectine 3, protĂ©ine impliquĂ©e dans lâactivation des macrophages ainsi que la rĂ©tention des LDL oxydĂ©es par ces derniĂšres. Le deuxiĂšme volet concerne une seconde approche de criblage in silico des anticorps issus de la sĂ©lection in vivo, notamment grĂące Ă lâutilisation de techniques de sĂ©quençage haut dĂ©bit (Pacific Biosciences). Les rĂ©sultats de cette Ă©tude pilote ont permis demettre en exergue plusieurs points : (1) la possibilitĂ© dâobtenir des sĂ©quences >1000bpcorrespondant Ă des scFv aprĂšs analyse par IMGT/HighV-QUEST (database des immunoglobulines internationales) et (2) la possibilitĂ© dâidentifier des sĂ©quences de clones prĂ©fĂ©rentiellement enrichies aprĂšs une sĂ©lection in vivo. En conclusion et perspectives, ces deux mĂ©thodes de criblage complĂ©mentaires nous donnent accĂšs Ă de nouvelles voies dâobtention dâanticorps humains pour (1) le couplage Ă des nanoparticules pour le diagnostic par IRM de la plaque dâathĂ©rome ainsi que (2) apporter une thĂ©rapie in situ au site inflammatoire.This thesis is based on the selection of human antibodies and identification of newbiomarkers to develop a diagnosis and a therapy of atherosclerosis. The part one treats, first,the selection of human antibodies in a pathophysiological context in an animal model ofatherosclerosis. To access not only to a large panel of targets but also proteins in their nativeconformation and influenced by their microenvironment, an in vivo selection was performed.In a second time, an innovative high throughput flow cytometry screening was implemented.Contrary to the use of ELISA screening which needs to have an identified target, the flowcytometry doesnât have this limitation and uses a lower quantity of proteins than ELISAassays. By this screening, two thousand clones were selected. Characterization and specificitytests of these antibodies have yielded to the discovery of galectin 3 has a new biomarker. Thisprotein is implicated in macrophage activation, binding of oxLDL by macrophages to formfoam cells. The second part is about a second screening approach of the antibodies issuingfrom the in vivo selection by using Next Generation Sequencing methods (PacificBiosciences). The results of this primary study allowed for highlighingt two points: (1) thefeasibility to obtain long reads >1000bp identified as scFv after analysis by IMGT/HighVQUEST(international database of immunglobulins) and (2) the feasibility to identify hits ofclones preferentially enriched during the in vivo selection. In conclusion and perspectives,these two complementary methods give the opportunity to obtain human antibodies for (1) thecoupling to nanoparticles for diagnosis by MRI of atheroma plaque and (2) serve as ligandsfor in situ delivery of drugs
A la recherche dâanticorps humains et de nouveaux biomarqueurs pour le ciblage de la plaque dâathĂ©rome
This thesis is based on the selection of human antibodies and identification of newbiomarkers to develop a diagnosis and a therapy of atherosclerosis. The part one treats, first,the selection of human antibodies in a pathophysiological context in an animal model ofatherosclerosis. To access not only to a large panel of targets but also proteins in their nativeconformation and influenced by their microenvironment, an in vivo selection was performed.In a second time, an innovative high throughput flow cytometry screening was implemented.Contrary to the use of ELISA screening which needs to have an identified target, the flowcytometry doesnât have this limitation and uses a lower quantity of proteins than ELISAassays. By this screening, two thousand clones were selected. Characterization and specificitytests of these antibodies have yielded to the discovery of galectin 3 has a new biomarker. Thisprotein is implicated in macrophage activation, binding of oxLDL by macrophages to formfoam cells. The second part is about a second screening approach of the antibodies issuingfrom the in vivo selection by using Next Generation Sequencing methods (PacificBiosciences). The results of this primary study allowed for highlighingt two points: (1) thefeasibility to obtain long reads >1000bp identified as scFv after analysis by IMGT/HighVQUEST(international database of immunglobulins) and (2) the feasibility to identify hits ofclones preferentially enriched during the in vivo selection. In conclusion and perspectives,these two complementary methods give the opportunity to obtain human antibodies for (1) thecoupling to nanoparticles for diagnosis by MRI of atheroma plaque and (2) serve as ligandsfor in situ delivery of drugs.Cette thĂšse porte sur la sĂ©lection dâanticorps humains et la dĂ©couverte de nouveaux biomarqueurs en vue dâun diagnostic et dâune thĂ©rapie de lâathĂ©rosclĂ©rose. Le premier volet concerne, dans un premier temps, la sĂ©lection dâanticorps humains dans un contexte physiopathologique chez un modĂšle animal de lâathĂ©rosclĂ©rose. La sĂ©lection in vivo a permis de sĂ©lectionner des anticorps sur large pannel de cibles dans leur conformation native et sous lâinfluence de leur microenvironnement. Suite Ă cela, afin dâaccĂ©der Ă la spĂ©cificitĂ© des clones, une mĂ©thode innovante de criblage a Ă©tĂ© dĂ©veloppĂ©e, la cytomĂ©trie de flux Ă moyen dĂ©bit. Cette technique, contrairement Ă lâELISA couramment utilisĂ© pour la caractĂ©risation dâanticorps ou de peptides, ne nĂ©cessite pas de connaitre lâantigĂšne et permet Ă©galement de travailler Ă des concentrations de protĂ©ines moindres. Ainsi, deux cents anticorps ont pu ĂȘtre sĂ©lectionnĂ©s. Les tests dâaffinitĂ© et de caractĂ©risation de ces anticorps ont, par la suite, menĂ© Ă lâidentification dâune nouvelle cible de la plaque dâathĂ©rome, la galectine 3, protĂ©ine impliquĂ©e dans lâactivation des macrophages ainsi que la rĂ©tention des LDL oxydĂ©es par ces derniĂšres. Le deuxiĂšme volet concerne une seconde approche de criblage in silico des anticorps issus de la sĂ©lection in vivo, notamment grĂące Ă lâutilisation de techniques de sĂ©quençage haut dĂ©bit (Pacific Biosciences). Les rĂ©sultats de cette Ă©tude pilote ont permis demettre en exergue plusieurs points : (1) la possibilitĂ© dâobtenir des sĂ©quences >1000bpcorrespondant Ă des scFv aprĂšs analyse par IMGT/HighV-QUEST (database des immunoglobulines internationales) et (2) la possibilitĂ© dâidentifier des sĂ©quences de clones prĂ©fĂ©rentiellement enrichies aprĂšs une sĂ©lection in vivo. En conclusion et perspectives, ces deux mĂ©thodes de criblage complĂ©mentaires nous donnent accĂšs Ă de nouvelles voies dâobtention dâanticorps humains pour (1) le couplage Ă des nanoparticules pour le diagnostic par IRM de la plaque dâathĂ©rome ainsi que (2) apporter une thĂ©rapie in situ au site inflammatoire
Glioblastoma Immune Landscape and the Potential of New Immunotherapies
International audienceGlioblastoma (GBM) are the most common tumors of the central nervous system and among the deadliest cancers in adults. GBM overall survival has not improved over the last decade despite optimization of therapeutic standard-of-care. While immune checkpoint inhibitors (ICI) have revolutionized cancer care, they unfortunately have little therapeutic success in GBM. Here, we elaborate on normal brain and GBM-associated immune landscapes. We describe the role of microglia and tumor-associated macrophages (TAMs) in immune suppression and highlight the impact of energy metabolism in immune evasion. We also describe the challenges and opportunities of immunotherapies in GBM and discuss new avenues based on harnessing the anti-tumor activity of myeloid cells, vaccines, chimeric antigen receptors (CAR)-T and -NK cells, oncolytic viruses, nanocarriers, and combination therapies
A nano-emulsion platform functionalized with a fully human scFv-Fc antibody for atheroma targeting: towards a theranostic approach to atherosclerosis
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 âtheranostic approachâ 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 Apoeâ/â 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.Translational Research and Advanced Imaging LaboratoryOptimization of therapeutic monoclonal antibodies development Better antibodies, better developed AND better use
An innovative flow cytometry method to screen human scFv-phages selected by in vivo phage-display in an animal model of atherosclerosis
Atherosclerosis is a chronic, progressive inflammatory disease that may develop into vulnerable lesions leading to thrombosis. This pathology is characterized by the deposition of lipids within the arterial wall and infiltration of immune cells leading to amplification of inflammation. Nowadays there is a rising interest to assess directly the molecular and cellular components that underlie the clinical condition of stroke and myocardial infarction. Single chain fragment variable (scFv)-phages issuing from a human combinatorial library were selected on the lesions induced in a rabbit model of atherosclerosis after three rounds of in vivo phage display. We further implemented a high-throughput flow cytometry method on rabbit protein extracts to individually test one thousand of scFv-phages. Two hundred and nine clones were retrieved on the basis of their specificity for atherosclerotic proteins. Immunohistochemistry assays confirmed the robustness of the designed cytometry protocol. Sequencing of candidates demonstrated their high diversity in VH and VL germline usage. The large number of candidates and their diversity open the way in the discovery of new biomarkers. Here, we successfully showed the capacity of combining in vivo phage display and high-throughput cytometry strategies to give new insights in in vivo targetable up-regulated biomarkers in atherosclerosis
In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis
BACKGROUND: Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid-laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment. METHODS AND RESULTS: Human scFv (single-chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co-immunoprecipitated with the selected scFv-Fc followed by mass spectrometry for target identification. Near-infrared fluorescence imaging was performed in Apoe â/â 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 nanoobjects 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 ÎČ-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 â/â 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
Multimodal molecular imaging of atherosclerosis: Nanoparticles functionalized with scFv fragments of an anti-αIIbÎČ3 antibody
Due to the wealth of actors involved in the development of atherosclerosis, molecular imaging based on the targeting of specific markers would substantiate the diagnosis of life-threatening atheroma plaques. To this end, TEG4 antibody is a promising candidate targeting the activated platelets (integrin αIIbÎČ3) 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 ApoEâ/â 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.DĂ©veloppment d'une infrastructure française distribuĂ©e coordonnĂ©
pPICZαA expression vector according to the EasySelect <i>Pichia</i> Expression Kit Manual (Invitrogen) and a schematic representation of TEG4-2c scFv with the protein sequence.
<p><b>(A)</b>: All the featured restriction sites are unique. 5Ž AOX1: promoter region of AOX1; TT AOX1: transcription termination of AOX1; PTEF1: promoter of TEF1; PEM7: promoter of EM7; Zeocin resistance: Sh ble ORF; CYC1 TT: transcription termination of CYC1. <b>(B)</b>: The TEG4-2c scFv coding sequence was cloned between <i>Pml</i>I and <i>Xba</i>I sites and the protein sequence of recombinant tag-scFv including the 6His-tag and the 2 cysteine are highlighted in green. The α-factor signal sequence is represented in blue and the C-myc tag is highlighted in yellow.</p
Binding assessment of TEG4-2c scFv to human platelets by flow cytometry and ELISA tests.
<p><b>A</b>: Binding of TEG4-2c scFv on thrombin-activated human (A-PL) or non-activatedâplatelets (NA-PL) analysed by flow cytometry. PAC-1 IgM murine antibody serves as a positive control. Binding of antibody to the platelets was further detected by incubation with Alexa Fluor 488 anti-6His or anti-mouse IgM antibodies. Negative controls were secondary antibody only. Histograms depict representative data ± SD of three independent experiments. Quantitative fluorescence intensities (in Geo mean) are stated under each respective histogram. <b>B</b>: Binding of TEG4-2c scFv on TRAP-activated-human (+ TRAP) or non-activated platelets (-TRAP) analysed by flow cytometry. Quantitative fluorescence intensities (in Geo mean) are stated under each respective histogram. <b>C</b>: Representative whole cell (A-PL, NA-PL) and purified proteins (αIIbÎČ3, BSA) ELISA with TEG4-2c scFv. A murine anti-αIIbÎČ3 antibody AP2 was used as positive control. Negative controls were secondary antibody only. Binding of antibodies was visualized via HRP-6His or HRP-anti-mouse IgG. OD value represents absorbance at 450 nm. Plots represent the mean values ± SD (n = 3)</p
TEG4-2c scFv production process. A: Fed batch fermentation history plot.
<p>Stirring, pO<sub>2</sub> and OD<sub>600</sub> values are plotted versus time during the cultivation of <i>P</i>. <i>pastoris</i> in BMGY medium. Cultures were induced with methanol at t = 0 (24 h after starting the batch phase) during the fed batch phase the methanol was added every 12 h or 6h (black arrows) to a final concentration of 0.6%. The average values are shown with error bars representing the standard deviation (n = 5). 1 OD<sub>600</sub> unit was equivalent to 0.29 mg/mL dry weight. <b>B: Dot-blot analysis of supernatants from recombinant <i>P</i>. <i>pastoris</i> culture</b>. Fifty microliters samples from non-induced culture (NI) and from day 1 to day 5 induced cultures (I1d to I5d) were undiluted (a) or diluted (b = 1:10; c = 1:50) and blotted on a nitrocellulose membrane. The recombinant TEG4-2c scFv were detected with the Anti-6His antibody and revealed by colorimetric analysis.</p