Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR
Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries an...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/21/15961 |
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| author | Anielka Zehnaker Amandine Vallet Juliette Gourdon Caterina Sarti Vinesh Jugnarain Maya Haj Hassan Laetitia Mathias Camille Gauthier Pauline Raynaud Thomas Boulo Linda Beauclair Yves Bigot Livio Casarini Pascale Crépieux Anne Poupon Benoît Piégu Frédéric Jean-Alphonse Gilles Bruneau Éric Reiter |
| author_facet | Anielka Zehnaker Amandine Vallet Juliette Gourdon Caterina Sarti Vinesh Jugnarain Maya Haj Hassan Laetitia Mathias Camille Gauthier Pauline Raynaud Thomas Boulo Linda Beauclair Yves Bigot Livio Casarini Pascale Crépieux Anne Poupon Benoît Piégu Frédéric Jean-Alphonse Gilles Bruneau Éric Reiter |
| author_sort | Anielka Zehnaker |
| collection | DOAJ |
| description | Developing modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR. |
| first_indexed | 2024-03-11T11:27:29Z |
| format | Article |
| id | doaj.art-80d2f09f88af4fa383e3ba0dc298aa0f |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T11:27:29Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-80d2f09f88af4fa383e3ba0dc298aa0f2023-11-10T15:05:56ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-11-0124211596110.3390/ijms242115961Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHRAnielka Zehnaker0Amandine Vallet1Juliette Gourdon2Caterina Sarti3Vinesh Jugnarain4Maya Haj Hassan5Laetitia Mathias6Camille Gauthier7Pauline Raynaud8Thomas Boulo9Linda Beauclair10Yves Bigot11Livio Casarini12Pascale Crépieux13Anne Poupon14Benoît Piégu15Frédéric Jean-Alphonse16Gilles Bruneau17Éric Reiter18Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FrancePhysiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche Pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS), Université de Tours, 37380 Nouzilly, FranceDeveloping modulatory antibodies against G protein-coupled receptors is challenging. In this study, we targeted the follicle-stimulating hormone receptor (FSHR), a significant regulator of reproduction, with variable domains of heavy chain-only antibodies (VHHs). We built two immune VHH libraries and submitted them to multiplexed phage display approaches. We used next-generation sequencing to identify 34 clusters of specifically enriched sequences that were functionally assessed in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. In this assay, 23 VHHs displayed negative or positive modulation of FSH-induced responses, suggesting a high success rate of the multiplexed strategy. We then focused on the largest cluster identified (i.e., PRC1) that displayed positive modulation of FSH action. We demonstrated that PRC1 specifically binds to the human FSHR and human FSHR/FSH complex while potentiating FSH-induced cAMP production and Gs recruitment. We conclude that the improved selection strategy reported here is effective for rapidly identifying functionally active VHHs and could be adapted to target other challenging membrane receptors. This study also led to the identification of PRC1, the first potential positive modulator VHH reported for the human FSHR.https://www.mdpi.com/1422-0067/24/21/15961phage displayVHHGPCRFSHRnext-generation sequencing |
| spellingShingle | Anielka Zehnaker Amandine Vallet Juliette Gourdon Caterina Sarti Vinesh Jugnarain Maya Haj Hassan Laetitia Mathias Camille Gauthier Pauline Raynaud Thomas Boulo Linda Beauclair Yves Bigot Livio Casarini Pascale Crépieux Anne Poupon Benoît Piégu Frédéric Jean-Alphonse Gilles Bruneau Éric Reiter Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR International Journal of Molecular Sciences phage display VHH GPCR FSHR next-generation sequencing |
| title | Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR |
| title_full | Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR |
| title_fullStr | Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR |
| title_full_unstemmed | Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR |
| title_short | Combined Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays as a Platform for Identifying Modulatory VHHs Targeting the FSHR |
| title_sort | combined multiplexed phage display high throughput sequencing and functional assays as a platform for identifying modulatory vhhs targeting the fshr |
| topic | phage display VHH GPCR FSHR next-generation sequencing |
| url | https://www.mdpi.com/1422-0067/24/21/15961 |
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