Screening, expression, and functional validation of camelid-derived nanobodies targeting RSPO2

IF 1.4 3区农林科学 Q4 IMMUNOLOGY

Veterinary immunology and immunopathology Pub Date : 2025-03-27 DOI:10.1016/j.vetimm.2025.110922

Shaojue Guo , Zhao Shuaiying , Kong Yingying , Junming Tang , Jianfeng Xu , Yuanyuan Dai , Yong Geng

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引用次数: 0

Abstract

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.

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来源期刊

Veterinary immunology and immunopathology 农林科学-免疫学

CiteScore

3.40

自引率

5.60%

发文量

审稿时长

70 days

期刊介绍： The journal reports basic, comparative and clinical immunology as they pertain to the animal species designated here: livestock, poultry, and fish species that are major food animals and companion animals such as cats, dogs, horses and camels, and wildlife species that act as reservoirs for food, companion or human infectious diseases, or as models for human disease. Rodent models of infectious diseases that are of importance in the animal species indicated above,when the disease requires a level of containment that is not readily available for larger animal experimentation (ABSL3), will be considered. Papers on rabbits, lizards, guinea pigs, badgers, armadillos, elephants, antelope, and buffalo will be reviewed if the research advances our fundamental understanding of immunology, or if they act as a reservoir of infectious disease for the primary animal species designated above, or for humans. Manuscripts employing other species will be reviewed if justified as fitting into the categories above. The following topics are appropriate: biology of cells and mechanisms of the immune system, immunochemistry, immunodeficiencies, immunodiagnosis, immunogenetics, immunopathology, immunology of infectious disease and tumors, immunoprophylaxis including vaccine development and delivery, immunological aspects of pregnancy including passive immunity, autoimmuity, neuroimmunology, and transplanatation immunology. Manuscripts that describe new genes and development of tools such as monoclonal antibodies are also of interest when part of a larger biological study. Studies employing extracts or constituents (plant extracts, feed additives or microbiome) must be sufficiently defined to be reproduced in other laboratories and also provide evidence for possible mechanisms and not simply show an effect on the immune system.