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Leucine-rich repeat-containing G-protein receptor 5 (LGR5) has been characterised as a stem cell and cancer stem cell marker. Previous analyses of LGR5 transcript levels indicate high level expression discriminates malignancies such as colorectal cancer (CRC) and pre-B acute lymphoblastic leukaemia (pre-B ALL) from healthy tissues suggesting LGR5 protein expression may provide a molecular handle for prognosis and treatment.We have developed highly specific, high affinity antibodies to the extracellular domain of human LGR5 (α-LGR5) that detect high LGR5 protein levels in colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL. In contrast, there is low to undetectable levels of LGR5 protein in normal colon and rectal epithelia, liver, ovarian tissues, brain and immune cell types.LGR5 is rapidly internalised from the plasma membrane and trafficked to intracellular vesicular compartments including lysosomes. Treatment of high LGR5-expressing CRC and pre-B ALL cancer cell lines with an antibody-drug conjugate version of α-LGR5 (α-LGR5-ADC) lead to effective cell killing at nanomolar concentrations. Interventional treatment of pre-B ALL tumours with α-LGR5-ADC in vivo led to rapid tumour attrition. We further demonstrated the therapeutic utility of humanised α-LGR5 by using the corresponding scFv fragment for the generation of α-LGR5 chimeric antigen receptors (CARs) and a Bispecific T cell Engager (BiTE). α-LGR5-CAR-NK cells were effective at killing LGR5-expressing cells while α-LGR5/α-CD3 BiTEs induce T cell activation and killing of NALM6 cells by cytotoxic CD8+ T cells.Taken together, this study establishes α-LGR5-based therapeutic modalities that effectively discriminate and target CRC, HCC and pre-B ALL tumour cells.One Sentence SummaryWe generated novel antibodies against the cancer cell marker LGR5, validated diagnostic use in prioritizing specific cancer types for targeting, and developed antibody-based therapeutics.