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Screening, expression, and functional validation of camelid-derived nanobodies targeting RSPO2
Shaojue Guo1, Zhao Shuaiying1, Kong Yingying2
1Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
Objective:
RSPO2 (R-spondin 2) is a key regulator of the Wnt/β-catenin signaling pathway, involved in embryogenesis, tissue homeostasis, and cancer progression. Despite its therapeutic potential, effective agents targeting RSPO2 remain elusive. To address the unmet need for RSPO2-targeted therapies, we aimed to develop high-affinity nanobodies via phage display and prokaryotic expression, characterizing their binding specificity and functional blockade of RSPO2-LGR4 interactions. This study provides foundational insights into nanobody-mediated inhibition of Wnt signaling, supporting future therapeutic strategies against RSPO2-driven pathologies.
Methods:
Recombinant RSPO2 proteins were constructed and purified using PCR-based recombination. Camels (Camelus bactrianus) were immunized with RSPO2, and phage display was employed to screen nanobody libraries. High-affinity nanobodies were cloned, expressed, purified, and assessed for specificity and binding affinity using biolayer interferometry and protein blotting. Functional validation was performed using TOPFLASH assays to evaluate their impact on Wnt/β-catenin signaling.
Results:
Nanobodies with high specificity and nanomolar-range affinity constants (KDs) for RSPO2 were identified. The nanobody effectively inhibited RSPO2-induced Wnt/β-catenin signaling in human renal epithelial cells.
Conclusion:
The development of RSPO2-targeting nanobodies offers new prospects for treating RSPO2-related diseases. The nanobody serve as valuable tools for functional research and hold potential as diagnostic and therapeutic agents for RSPO2-driven conditions.
Insights
Researchers developed high-affinity nanobodies targeting R-spondin 2 (RSPO2) to inhibit Wnt/β-catenin signaling. These nanobodies show promise for treating RSPO2-driven diseases and advancing therapeutic strategies.
Area of Science:
- Molecular Biology
- Immunology
- Cancer Biology
Background:
- R-spondin 2 (RSPO2) is a critical regulator of the Wnt/β-catenin pathway, implicated in development, tissue maintenance, and cancer.
- Targeted therapies for RSPO2 are lacking, representing a significant unmet medical need.
- Understanding RSPO2's role is crucial for developing novel therapeutic interventions.
Purpose of the Study:
- To develop high-affinity nanobodies against RSPO2 using phage display.
- To characterize the binding specificity and functional inhibitory capacity of these nanobodies.
- To explore the potential of nanobody-mediated Wnt pathway inhibition for therapeutic applications.
Main Methods:
- Recombinant RSPO2 protein production and purification.
- Immunization of camels and construction of phage display nanobody libraries.
- Screening, cloning, expression, and purification of high-affinity nanobodies.
- Assessment of binding affinity (biolayer interferometry) and specificity (protein blotting).
- Functional validation of Wnt/β-catenin signaling inhibition (TOPFLASH assays).
Main Results:
- Identification of nanobodies with high specificity and nanomolar affinity for RSPO2.
- Demonstration of effective inhibition of RSPO2-induced Wnt/β-catenin signaling in human renal epithelial cells.
Conclusions:
- Development of novel RSPO2-targeting nanobodies presents new therapeutic avenues.
- These nanobodies are valuable tools for research into RSPO2-driven pathologies.
- Potential applications include diagnostics and therapeutics for RSPO2-related diseases.
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