Research Fellowship in Antibody fragments targeting ovarian GPCRs to control reproduction

In this study, we tackled the challenge of developing modulatory antibodies against G protein-coupled receptors, with a specific focus on the follicle-stimulating hormone receptor (FSHR), a pivotal regulator of reproduction. Leveraging variable domains of heavy chain-only antibodies (VHHs), we constructed two immune VHH libraries and implemented multiplexed phage display techniques. Our methodology integrated Multiplexed Phage Display, High-Throughput Sequencing, and Functional Assays to identify modulatory VHHs targeting FSHR. Following library construction, next-generation sequencing identified 34 clusters of specifically enriched sequences. These sequences underwent functional assessment in a primary screen based on a cAMP response element (CRE)-dependent reporter gene assay. Impressively, 23 VHHs displayed either negative or positive modulation of FSH-induced responses, indicating a high success rate for the multiplexed strategy. Subsequently, we focused on the largest identified cluster, PRC1, which exhibited positive modulation of FSH action. We provided evidence that PRC1 specifically binds to human FSHR and the FSHR/FSH complex, enhancing FSH-induced cAMP production and Gs recruitment. In conclusion, our study showcases an improved selection strategy that effectively identifies functionally active VHHs and can be adapted to target other challenging membrane receptors. Notably, this investigation led to the discovery of PRC1, the first potential positive modulator VHH reported for the human FSHR.