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...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2021-10-01
|
| Series: | Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease |
| Subjects: | |
| Online Access: | https://www.ahajournals.org/doi/10.1161/JAHA.120.016287 |
| _version_ | 1797936076392759296 |
|---|---|
| author | Audrey Hemadou Alexandre Fontayne Jeanny Laroche‐Traineau Florence Ottones Philippe Mondon Stéphane Claverol Éric Ducasse Stéphane Sanchez Sarah Mohamad Cyril Lorenzato Martine Duonor‐Cerutti Gisèle Clofent‐Sanchez Marie‐Josée Jacobin‐Valat |
| author_facet | Audrey Hemadou Alexandre Fontayne Jeanny Laroche‐Traineau Florence Ottones Philippe Mondon Stéphane Claverol Éric Ducasse Stéphane Sanchez Sarah Mohamad Cyril Lorenzato Martine Duonor‐Cerutti Gisèle Clofent‐Sanchez Marie‐Josée Jacobin‐Valat |
| author_sort | Audrey Hemadou |
| collection | DOAJ |
| description | 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 β‐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. |
| first_indexed | 2024-04-10T18:24:03Z |
| format | Article |
| id | doaj.art-02099d21a4c148fcb3e8f0b52035004b |
| institution | Directory Open Access Journal |
| issn | 2047-9980 |
| language | English |
| last_indexed | 2024-04-10T18:24:03Z |
| publishDate | 2021-10-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease |
| spelling | doaj.art-02099d21a4c148fcb3e8f0b52035004b2023-02-02T06:18:44ZengWileyJournal of the American Heart Association: Cardiovascular and Cerebrovascular Disease2047-99802021-10-01101910.1161/JAHA.120.016287In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of AtherosclerosisAudrey Hemadou0Alexandre Fontayne1Jeanny Laroche‐Traineau2Florence Ottones3Philippe Mondon4Stéphane Claverol5Éric Ducasse6Stéphane Sanchez7Sarah Mohamad8Cyril Lorenzato9Martine Duonor‐Cerutti10Gisèle Clofent‐Sanchez11Marie‐Josée Jacobin‐Valat12CRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceLFB (Laboratoire Français de Fractionnement et de Biotechnologies) Biotechnologies Lille FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceLFB (Laboratoire Français de Fractionnement et de Biotechnologies) Biotechnologies Lille FranceProtéome Pole CGFB (Centre de Génomique Fonctionnelle de Bordeaux) Bordeaux FranceCHU Pellegrin Bordeaux FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceUPS3044CNRS (Centre National de Recherche Scientifique) Saint‐Christol‐Lès‐Alès FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceCRMSB (Centre de Resonance Magnétique des Systèmes Biologiques)UMR5536 CNRS (Centre National de Recherche Scientifique)INSB (Institut National des Sciences Biologiques) Bordeaux FranceBackground 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 β‐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.https://www.ahajournals.org/doi/10.1161/JAHA.120.016287biomarkersflow cytometryhuman antibodiesimagingin vivo phage display |
| spellingShingle | Audrey Hemadou Alexandre Fontayne Jeanny Laroche‐Traineau Florence Ottones Philippe Mondon Stéphane Claverol Éric Ducasse Stéphane Sanchez Sarah Mohamad Cyril Lorenzato Martine Duonor‐Cerutti Gisèle Clofent‐Sanchez Marie‐Josée Jacobin‐Valat In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease biomarkers flow cytometry human antibodies imaging in vivo phage display |
| title | In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis |
| title_full | In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis |
| title_fullStr | In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis |
| title_full_unstemmed | In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis |
| title_short | In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis |
| title_sort | in vivo human single chain fragment variable phage display assisted identification of galectin 3 as a new biomarker of atherosclerosis |
| topic | biomarkers flow cytometry human antibodies imaging in vivo phage display |
| url | https://www.ahajournals.org/doi/10.1161/JAHA.120.016287 |
| work_keys_str_mv | AT audreyhemadou invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT alexandrefontayne invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT jeannylarochetraineau invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT florenceottones invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT philippemondon invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT stephaneclaverol invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT ericducasse invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT stephanesanchez invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT sarahmohamad invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT cyrillorenzato invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT martineduonorcerutti invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT giseleclofentsanchez invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis AT mariejoseejacobinvalat invivohumansinglechainfragmentvariablephagedisplayassistedidentificationofgalectin3asanewbiomarkerofatherosclerosis |