The incidence of early-onset colorectal cancer (CRC) is increasing worldwide. To address this challenge, we have developed a CAR-T cell therapy (CNA3103), targeting leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), which is currently undergoing a first-in-human Phase I/IIa clinical trial (NCT05759728). LGR5 is known to potentiate Wnt/β-catenin signalling and mark cells-of-origin in intestinal cancers. Extensive screening demonstrated significantly elevated expression of Lgr5 in CRC, which is correlated with reduced patient survival. In CRC, LGR5 has been intimately linked with metastasis, cell survival and perturbation of chemotherapeutic inventions. Utilizing an optimised 9-day preclinical manufacturing method, LGR5-targeting CAR-T cells were generated with minimally differentiated T cell phenotypes, which was concomitant with significant cytotoxic and activation marker expression. In human CRC xenograft mouse models, LGR5-targeting CAR-T cells eradicated tumours at doses as low as 800,000 CAR+ cells, and were capable of significantly inhibiting advanced tumour growth following delayed administration. Robust CAR-T cell-mediated immunological protection was observed in rechallenge experiments, in which secondary tumour growth was undetectable in mice which had tumours rejected following previous CAR-T cell administration. LGR5 protein and mRNA expression was also detected in a diverse range of cancer families, including liver, ovarian, brain and stomach, expanding upon the clinical indications with which LGR5-targeting CAR-T cells may be harnessed. CNA3103 is currently undergoing dose escalation in a Phase I/IIa clinical trial in advanced metastatic colorectal cancer patients, showing very limited toxicity and evidence of CAR-T cell engraftment.