Abstract
BACKGROUND: Antibody-drug conjugates (ADCs) are a significant advancement in targeted cancer therapy, but none are approved for colorectal cancer (CRC). LGR4/5/6, highly expressed in most CRCs, are promising targets. While LGR5-targeting ADCs show strong anti-tumor effects, their efficacy is limited by LGR5 loss in some CRC cells. RSPO4, a natural ligand for LGR4/5/6, binds all three receptors with high affinity. This research develops RSPO4-based peptibody drug-conjugates (PDCs) to simultaneously target LGR4/5/6, offering a novel therapeutic approach for CRC. METHODS: LGR4/5/6 expression in CRCs was analysed using RNA-seq datasets and Western blot. Peptibody binding affinities were measured, conjugated to camptothecin analog, CPT2, and tested for cytotoxicity in CRC cell lines. Antitumor efficacy was evaluated in vivo using CRC cell line and patient-derived xenograft (PDX) models. RESULTS: Peptibody was engineered by fusing a mutant RSPO4 furin-domain to human IgG1 Fc, retaining high-affinity LGR4/5/6 binding without enhancing Wnt/β-catenin signalling. Conjugated with CPT2 molecules, the PDC showed strong antitumor activity in CRC cell lines and dose-dependent tumor growth inhibition in xenograft and patient-derived models. CONCLUSION: Preclinical data showed that LGR4/5/6-targeting PDC exhibited potent cytotoxicity in vitro and robust antitumor efficacy in CRC xenograft and PDX models, making its potential as a promising therapeutic approach for CRC.