Supplementary Fig. S1- Coomassie gel of anti-LGR5 mAb and anti-LGR5 ADCs. Supplementary Fig. S2- Confocal image of HEK293T cells with stable over-expression of hLGR4. Supplementary Fig. S3- Anti-LGR5 ADC binding assay and cytotoxicity of free MMAE. Supplementary Fig. S4- Anti-LGR5 mAb crossreactivity with mouse LGR5 and images of intestinal organoids treated with anti-LGR5 ADC or free MMAE. Supplementary Fig. S5- Representative images of LoVo xenograft tumors in mice from each treatment cohort and graph of average body weight. Supplementary Fig. S6- H&E staining of intestinal tissue sections. Supplementary Fig. S7. Images of Lovo xenograft tumor recurrence and weight.
ARTICLE ABSTRACTGastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein–coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody–drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580–90. ©2016 AACR.